Conserving genetic variation in British populations of Lobelia urens

PII:

S0006-3207(96)00115-2

Biological Conservation, 79 (1997) 15 22 Copyright © 1996 Elsevier Science Limited Printed in Great Britain. All rights reserved 0006-3207/97 $17.00 + .00

ELSEVIER

C O N S E R V I N G G E N E T I C V A R I A T I O N IN BRITISH P O P U L A T I O N S OF L o b e l i a u r e n s R. E. Daniels, a A. F. R a y b o u l & & J. M. F a r k a s b alnstitute of Terrestrial Ecology, Furzebrook Research Station, BH20 5AS, Wareham, UK bDivision of Environmental Sciences, University of Hertfordshire, Hatfield Campus, College Lane, ALIO 9AB, Hatfield, UK

(Received 24 February 1996; accepted 24 June 1996)

Abstract

Several models have been proposed to explain how genetic diversity is influenced by population size and degree of isolation, and by population history, operating through processes such as founder effects, genetic drift and bottlenecks (e.g. Wright, 1931; Lacy, 1987; Nei, 1975). However, as noted by Barrett and Kohn (1991), most of this theoretical work on stochastic loss of variation has been developed using outbreeding organisms with separate sexes and so may not be readily applicable to plants with hermaphrodite flowers and at least some capacity for self-fertilisation. The problems of assessing effects attributable to genetic drift or bottlenecks on theoretical grounds from observations of the genetic structure of current populations, will contain a number of potential sources of error, such as the extent to which self-pollination occurs, compatibility relationships among members of the population, individual longevity and fertility and seed bank dynamics. Few cases exist where there has been the opportunity to test the models predicting changes in allele frequency or heterozygosity in field populations of perennial plants. One example is that of pitcher-plant Sarracenia purpurea in Ireland, where several new secondary populations were established following introduction of the species from North America to Termonbarry Bog in 1906. Taggart et al. (1990) found that, as predicted by Nei (1975), severe bottlenecks (founder events involving 2 4 individuals) had a marked effect in reducing the number of polymorphic loci but produced only a small reduction in heterozygosity among the genetically differentiated daughter populations. Heath lobelia Lobelia urens L. (Campanulaceae) is a perennial species which, in Britain, occurs only at a few disjunct localities in southern England. Present populations differ considerably in size and their recent history is well documented (Dinsdale et al., in press), making this species ideal for investigations of some problems in conservation genetics. Because L. urens has only ever been recorded from a few isolated sites in Britain, reintroduction to former sites is not a practical option for maximising its chances of survival. Conservation of the species and its full range of genetic diversity must, therefore, be concentrated on appropriate management

Conservation of genetic variation in a species which occurs as a few disjunct populations poses particular problems related to both the separation of those populations and their size. Lobelia urens is rare in Britain and occurs in only six widely separated populations. The distribution of genetic variation within and among these populations was examined using enzyme electrophoresis. The results showed that much of the variation was held among populations ( G s r = 0.265) and that gene flow among populations was low. The populations therefore require conservation as independent units. No link was found between geographical distance and genetic distance, adding weight to the theory that the British populations were established independently. Evidence was found that the smaller, unmanaged, populations contained less variation, although all had passed through a series o f bottlenecks during the past 50 years. As surrounding vegetation becomes more dense, fewer individuals contribute to variation in population size and it is suggested that periodic disturbance should be used as a management tool in order to release variation accumulated in the seed bank and so prevent depletion of genetic diversity. Copyright © 1996 Elsevier Science Limited Keywords: genetic structure, isolation, management, isozymes.

INTRODUCTION The need to conserve genetic variation in rare or threatened species has been recognised by several authors (Frankel & Soulf, 1981; Simberloff, 1988; Falk & Holsinger, 1991; Barrett & Kohn, 1991). Loss of variation may be especially acute among species with small, isolated populations, when genetic drift and inbreeding become increasingly important processes (Lacy, 1987; Barrett & Kohn, 1991; Ellstrand & Elam, 1993; Raijmann et al., 1994). In small plant populations, large and unpredictable changes in allele frequency may occur due to drift (Barrett & Kohn, 1991; Ellstrand & Elam, 1993), resulting in an increased opportunity for loss of rare or uncommon alleles. 15

16

R. E. Daniels et al.

of existing populations. Can information on the genetic structure of these populations help us design appropriate management programmes to achieve these conservation objectives? Isozymes may be used as neutral markers to investigate population processes which, together with information on plant demography, are necessary precursors to examination of the consequences of non-neutral effects of importance in determining fitness and survival. Our objectives in the present study were to determine the amount of genetic variation within and among the British populations of L. urens, and to examine the effects of population size, isolation and history on the partitioning of this variation, as a guide to the likely genetic consequences of future changes in population characteristics resulting from different management practices.

minated seed may remain viable in the soil for many years. The species has a range extending from North Africa, the Azores and Madeira to southern England and it is declining over much of this range, including its distributional centre in western France (J.-M. Gehu, pers. comm.). The decline in abundance appears to be largely the product of changes in land use and the resulting loss of suitable habitat conditions for its survival. In the southern part of its range, the species is found predominantly in wet meadows and wet heath, though in the north it also occurs as a component of drier acid grassland and heath communities. The six extant British populations are scattered along the southern coastal counties of England from Cornwall to Sussex (Fig. 1). Although most of these populations were formerly either more extensive or formed part of local population groups, there is little evidence that the species was ever widespread or common. It was first noted at Shute Common near Axminster, Devon in 1778 (Clarke, 1778) and has been recorded from 19 locations in southern England, most of which represent sporadic occurrences or short-lived introductions (Dinsdale, 1996). The sampled populations show differences in size, extent, habitat conditions and management history, as summarised below (Bates, 1992; Dinsdale, 1996; pers. obs.).

THE SPECIES A N D ITS BRITISH POPULATIONS Lobelia urens is a perennial herb with overwintering buds which give rise to rosettes of leaves and erect flowering stems the following year (Brightmore, 1968). The longevity of individuals in the field is not known. Flowering usually begins in July and continues through into the autumn. The protandrous flowers are visited mainly by flies and out-breeding is the norm (Brightmore, 1968). However, observations by us on individual, isolated, plants show that production of viable seed by selfing can occur. Although seeds have no specialised dispersal structures, they are small and light, so that dispersal over a moderate distance is possible. L. urens seems to be favoured by disturbance and shows several characteristics of arable weeds, such as lack of a need for after-ripening of seed, rapid germination favoured by light and high or fluctuating temperature, and a capacity to bank seed (Dinsdale, 1996). Although ripe seed may germinate soon after shedding, all but the earliest seedlings fail to reach a sufficiently large size to overwinter successfully (Dinsdale, 1996). Unger-

r

j

I

ICalmin~ton ....

Redlake Cottage Meadows, Cornwall (grid ref. S X 126592). This population is present in two adjacent wet meadows with a history of use for rough grazing. The number of individuals present has fluctuated considerably over the past 30 years. There has been a particularly dramatic reduction over the past 10 years, especially following the introduction of Exmoor ponies in 1993. Andrew's Wood, Devon (grid ref. S X 710515). This large population (averaging more than 2000 plants) is distributed among several compartments of cleared woodland and acid grassland. It is part of a once very extensive tract of grassland and heath that contained

Irant°n

_I-~_rst Admiral' ~ ___

•

•

,

Wood

2o

Fig. 1. Locations of Lobelia urens populations sampled.

Conserving variation in Lobelia plants of L. urens. Different compartments of the site have different land use histories, including different grazing regimes and dates of woodland clearance. Kilmington, Devon (grid ref. S Y 252987). A small population of fewer than 50 plants (the only one remaining of four recorded in the vicinity) in improved grassland along a woodland edge. Current plant numbers are similar to those recorded 30 years ago Hurst Heath, Dorset (grid ref. S Y 784896). This large population (c. 2000 plants) is confined in a wet heath clearing in Pinus sylvestris/Betula pubescens woodland. Over the past 40 years the population has increased from a few scattered individuals to its present size largely as a result of vegetation clearance and subsequent 'active' management to encourage the plants. Hinton Admiral, Hampshire (grid ref . S Z 205951). This population of fewer than 100 plants has shown considerable fluctuation in numbers over the past 50 years. It is now restricted to a small ditch and the edge of a footpath in damp grass heath. Flimwell, Sussex (grid ref. TQ 722307). A large population (> 2000 plants) is present inside a wildfowl park and around the cleared woodland margins along the outer part of its fence line. It apparently developed from a few remaining individuals present along coppice rides before tree felling some 10 years ago. Morphometric measurements in a half-sib family trial using seed collected at three of the sites (Andrew's Wood, Hurst Heath and Hinton Admiral) suggested that amongfamily variation was more significant than betweenpopulation variation in the two larger populations but that Hinton Admiral was distinctive (Daniels, 1990).

METHODS Electrophoresis

In June 1994, leaves were collected from all plants in small populations and from a random selection of plants in all compartments of the large populations. In each case a young mature leaf was cut from its parent plant and placed in a labelled polythene bag which was stored temporarily in a cool box together with two freezer blocks. The samples were placed in a deep freeze (-73°C) within 36 h of collection and stored until analysed in 1995. Enzyme extracts were prepared by grinding individual leaves (c. 30 mg of tissue) in 400 /zl of 0.1 M Tris-HC1 buffer (pH 7.0) containing 10% glycerol, 1% ascorbic acid and 0.1% mercaptoethanol. The resulting slurry was centrifuged at 14,000 rpm for 3 min and the supernatant was stored at -73°C ready for electrophoresis. Vertical polyacrylamide gel electrophoresis was performed using 1-mm thick gels in a BioRad Protean II

17

electrophoresis chamber. A 13% separating gel was prepared using a 0.4 M Tris-HC1 buffer, pH 8.8 and a 7% stacking gel prepared in 0.1 M Tris-HC1, pH 6.8. The electrode buffer was 0.072 M glycine-0.005 M Tris, pH 8.5. Approximately 50 ~tl of sample was used and electrophoresis was carried out at a constant 250 V for 7 h. Gels were stained for Acid Phosphatase (ACP), Glutamate Oxaloacetate Transaminase (GOT), Phosphoglucose Isomerase (PGI) and Esterase (EST) using the Raybould et al. (1991) modifications of the recipes of Shaw and Prasad (1970). Statistical methods

The total number of alleles was recorded and provided the basis for estimating genetic variation in terms of observed heterozygosity (Ho) and genetic diversity (Ht) (Nei, 1973) for both individual loci and means over all loci. The degree of population non-random mating was assessed by calculating Fis values ( f of Weir & Cockerham, 1984) and testing their significance using the computer program FSTAT (Goudet, 1995). The genetic differences among sites were examined by calculating Nei's genetic distance (D) (Nei, 1972) between all pairs of populations using the computer program Gendist in PHYLIP 3.4 (Felsenstein, 1989) and plotting them against the corresponding geographic distance (Singh & Rhomberg, 1987). As all pairwise genetic distances are not independent of each other, the significance of the correlation between genetic and geographic distances was estimated by a Mantel test (Mantel, 1967) as described by Raybould et al. (1996b). This test randomly reorders populations for calculation of genetic distance, while keeping populations in the same order for geographic distance, and calculates a new correlation coefficient for each permutation. For a positive correlation the significance of the correlation coefficient is the proportion of randomly derived correlation coefficients which are greater than, or equal to, the observed coefficient. The amount of gene flow (Nm) between all pairs of populations was estimated from the relationship Nm = (1/4Fst) - 1/4 (where Fst is the contribution that subpopulation differences make to the overall inbreeding coefficient, Wright, 1943). Because the mean Fis of all populations was significant (and hence Fst between these populations would be overestimated), Fst between pairs of populations was calculated using the procedure of Raybould and Mogg (in press). Briefly, the populations were subdivided into smaller groups, on the basis of the plants' spatial distributions, and the mean Fis of all groups was calculated. The procedure was repeated until the mean Fis of the groups was non-significant (Goudet et al., 1994; Raybould et al., 1996a). Fst between a pair of populations was then calculated as the mean of all pairwise Fsts between groups in different populations. Nm was then derived from this mean Fst. In order to examine how past and present demographic processes (such as population bottlenecks) may have influenced allelic variation, we obtained data on

R. E. Daniels et al.

18

the minimum (since 1945) and present (1994) population sizes for the six populations from Dinsdale et al. (in press) and Bates (1992). We then carried out rank correlations of Ht and number of alleles with these population sizes. We also calculated the rank correlation with our sample size to see whether sampling error may have influenced the results.

RESULTS Recent population history

All the populations present in Britain appear to have gone through one or more bottlenecks within the past 50 years, but since the early 1960s the patterns of population growth and decline have differed (Fig. 2). Only at Kilmington have numbers remained consistently low. The Andrew's Wood population has

shown a series of sharp fluctuations between 1000 and 5500 plants, though numbers fell below 200 in 1973. Similar sharp fluctuations occurred in populations at Redlake and Hinton Admiral though both the maximum and minimum number of plants were less. In contrast, the Flimwell population remained small until 1986, after which it grew rapidly. The same situation occurred at Hurst Heath where the number of plants has been kept high through active management (rotovation of different parts of the area on a rotational basis). The historical increases in population size at these sites can be related to episodes of either woodland clearance or the introduction of grazing, with its attendant trampling, and the creation of conditions under which seed either from any remaining individuals or from the persistent seed bank can germinate readily. As vegetation height and density increase, further germination is precluded and, as existing plants die, the population declines.

1400 ] - •- Kilmington

1200

• Redlake -"~- Hinton Admiral

~ 1000: •.~

800

0

,~ 600

~

/,

400 200

\ 'l~"~l~

•

o~ _'z" i-

"r - i - p

I

Year

6000 1~ I ~

/~

5000

~, 1\

[ " •" Flimwell ] "_H~uudrtHSeaW°°d

'~t - 4000 O

~ 3000 0

~ 2000

1

'.--., /

IOOO 0

Year Fig. 2. Changes in the population size of Lobelia urens at the six sample sites since 1963.

Conserving variation in Lobelia Allele and genotype frequencies Table 1 shows allele frequencies for individual populations and for the full set of samples. Different patterns of variation are shown by the loci measured, so that whilst there is only limited variation at GOT-l, other loci show distinct deviations from the overall mean allele frequency in some populations. This is particularly noticeable in fixation of the PGI-lb allele in the Kilmington and Hinton Admiral populations. The genetic structure of L o b e l i a populations The degree of non-random mating within each population is given as Fis (Table 2). The significance of Fis was tested using a randomisation procedure (Goudet, 1995). It can be seen that there is wide variation among the populations. Andrew's Wood, Hinton Admiral and Flimwell have significant Fis, while Redlake, Hurst Heath and Kilmington have significant heterozygote excesses. The Hinton Admiral population occupies a very small area (c. 5 m × 20 m) and perhaps, therefore, the heterozygote deficit is unlikely to be due to substructure. Unfortunately this could not be confirmed as the spatial distribution of the plants was not recorded. Flimwell and Andrew's W o o d are larger populations and at these sites the heterozygote deficit may be due to genetic substructuring of the population. To test this the two populations were subdivided on the basis of the spatial distribution of the plants. Andrew's W o o d was divided into the six management compartments (see section 2) and Flimwell into three groups. The mean Fis of the groups over all loci was obtained and tested as

19

before. F o r Andrew's W o o d mean group Fis was 0.174 (P (Fis ;~0) =0.008) and for Flimwell was 0.112 (P (Fis 50) =0.115). As mean group Fis is not significant at Flimwell, it appears that the significant Fis for the whole population is due to substructuring. At Andrew's W o o d the compartments give a significant mean Fis, suggesting inbreeding as a cause of high Fis in the whole population. However, the individual group or compartment Fis values vary widely within each site. Thus it seems likely that the processes causing n o n - r a n d o m mating vary within the larger sites and it is difficult to generalise about whether the significant whole population Fis arises from inbreeding or substructure. The number of alleles, H t and H o also show wide variation a m o n g the populations (Table 2). As may be expected from the Fis, the difference between Ht and H o is also very variable.

Population size relationships There was no correlation between sample size and either the diversity index or number of alleles (Table 3), indicating that variation among sites was not due to sampling error. Also there was no correlation of genetic variation with minimum population size over the past 50 years. This suggests that small numbers of plants are not indicative of a genetic bottleneck, probably because the effective population sizes are maintained at a higher level because of the presence of a seed bank. There was a strong correlation between 1994 population size and numbers of alleles detected. This relationship was also

Table 1. Allele frequencies in British populations of Locus

GOT- 1 PGI-I EST- I AcP- I

Allele

Slow Medium Fast Slow Medium Fast Slow Fast Slow Fast

L o b e l i a urens

Population a Redl

And

Kilm

Hurst

Hint

Flim

All sites

0.750 0.000 0.250 0.025 0.250 0.725 0.423 0.577 0.500 0.500

0.525 0.008 0.467 0.410 0.157 0.433 0.267 0.833 0.789 0.211

0.800 0.000 0.200 0.000 1.000 0.000 0.077 0.923 0.182 0.818

0.971 0.000 0.029 0.519 0.006 0.475 0.325 0.675 0.509 0.491

0.654 0.000 0.346 0.000 1.000 0.000 0.074 0.926 0.949 0.051

0.806 0.042 0.153 0.295 0.577 0.128 0.059 0.941 0.806 0.194

0.751 0.009 0.240 0.307 0.365 0.328 0.175 0.825 0.697 0.303

"Redl, Redlake; And, Andrew's Wood; Kilm, Kilmington; Hurst, Hurst Heath; Hint, Hinton Admiral; Flim, Flimwell.

Table 2. Summary of the genetic data for the L o b e l i a Population (sample size) Redlake (20) Andrew's Wood (73) Kilmington (18) Hurst Heath (91) Hinton Admiral (45) Fiimwell (39)

urens

populations

Total number of alleles

Mean Ht

Mean Ho

Fis

P (Fis~O)

P (Fis 7kO)

9 10 7 9 7 10

0.444 0.427 0.190 0.376 0.165 0.333

0.635 0.302 0.230 0.420 0.111 0.240

-0.378 0.279 -0.156 -0.097 0.339 0.302

0.996 < 0.001 0.999 0.950 0.003 < 0.001

0.005 > 0.999 0.475 0.050 0.999 > 0.999

20

R . E. D a n i e l s

et al.

Table 3. Rank correlations between measures of allelic diversity in

Diversity Index (Ht) Number of alleles

L o b e l i a urens

and population size

Sample size

Minimum population size

Present population size

rs = 0.143 NS rs = 0.359 NS

rs = 0.257 NS rs = -0.129 NS

rs = 0.543 NS rs = 0.951"*

Significance of rs (one-tailed) from Siegel (1956). NS not significant, **p < 0.01. significant at the 5% level following a sequential Bonferroni table-wide test (Rice, 1989). One interpretation is that 1994 population sizes were, at least in part, related to the management strategies at the sites, and that it was the different managements that affected the allelic variation in the populations. Genetic distance and gene flow among populations

Figure 3 shows a plot of all pairwise genetic distances (D) between the L o b e l i a populations against the corresponding geographic distance. The correlation coefficient between D and geographic distance is -0.416, suggesting that the genetic distance between two populations is actually reduced the further apart they are. However, a Mantel test indicated that the correlation is not significant (p=0.0758 with 10,000 randomisations), suggesting that there is no relationship between genetic and geographic distances. Inferred rates of gene flow between populations were found to be low (Table 4). In all but one case pairwise Nm values were < I. As expected from the correlation of genetic and geographic distances, Slatkin's (Slatkin, 1993) test for isolation by distance by a log-log regression of Nm and distance was not significant (b=0.364, r 2 = O. 168, Mantel test probability for a positive regression = 0.123). A significant negative relationship is expected with isolation by distance.

DISCUSSION If the populations found today represent remnants of a formerly more widespread occurrence, then we might 0.45

q~

"¢::1 ~J

0.35

--

00

expect to find a positive relationship between genetic distance between the populations and their geographical distance. Because no evidence was found for such a relationship, the implication is that the populations have arisen independently. Historical evidence supports this suggestion, as records show that the species has always had a scattered distribution. However, genetic distance estimates are biased by non-random mating within populations. The nature of the bias depends upon the cause of the non-random mating. Among inbred populations D is overestimated, while among structured populations it is underestimated (C. Gliddon, pers. comm.). As discussed above, there may be both inbreeding and substructure in the L o b e l i a populations which have significant Fis and thus it is possible that these biases may cancel out. Whatever the nature of the biases it seems highly unlikely that a significant positive relationship would result from unbiased estimates of D. Assuming the isozymes to be neutral markers this result is consistent with independent origins for the U K populations with insufficient subsequent gene flow to have significantly reduced the initial allele frequency differences (see below). A substantial amount of allelic variation is held among populations in L . urens. Over all loci GST was 0.265, which is just outside the upper 95% confidence interval for the mean GST value for animal-pollinated plants with mixed mating systems (Hamrick & Godt, 1989). This suggests that some selfing may occur in the field (species classed as selfers have a mean GST of 0.510+0.069). The L . urens GST value is also in the range for species with a narrow geographic range (0.242 4-0.047) (Hamrick & Godt, 1989). The amount of population differentiation as measured by Gsx at isozyme loci is higher than that as measured by analysis of seed protein profiles by Shannon's diversity index (King & Schaal, 1989). Analysis of the data in Table 3 of

- -

0.25

--

0.15

--

Table 4. Estimates rates of gene flow (Nm) between urens populations

RL z 0.05

-I

I

1 O0

200

Geographic distance (kin) Fig. 3. Relationship between genetic distance and geographic

distance for British Lobelia urens populations.

RL AW K HH HA FL

AW

K

HH

HA

Lobelia

FL

0.80219 -0.40003 0.26419 -1.104280.66777 0.19030 -0.29837 0.30183 0.26829 0.18427 -0.54315 0.57082 0.42428 0.37630 0.82493

RL, Redlake; AW, Andrew's Wood; K, Kilmington; HH, Hurst Heath; HA, Hinton Admiral; FL, Flimwell.

Conserving variation in Lobelia

Daniels (1990) shows that 16.8% of seed protein variation is held among populations. However, this diversity measure was calculated by treating each banding position as a separate phenotypic class. In reality, not all bands will be independent. If multibanded phenotyes are scored, inevitably the number of phenotypic classes that show no differentiation among populations is reduced. Thus 16.8% is the minimum proportion of seed protein variation held among populations in L. urens. Assuming that the populations have reached equilibrium (Takahata, 1983), then the amounts of gene flow (Table 4) are too low to prevent further differentiation through drift (Slatkin, 1994). Thus the amount of allelic variation held among populations (GsT) may increase from its already high value. This being so, all populations of L. urens have high conservation value, at least in terms of ailelic variation at isozyme loci. If isozyme loci are good indicators of variation in other parts of the genome then management practices that promote variation at isozyme loci will be of general conservation value. Inspection of the genetic variation within individual populations along with their management practices allows us to make suggestions of how variation within L. urens in the Bristish Isles may best be conserved. The presence of 10 alleles in the two largest populations, compared with seven for the two smallest, gives some indication of the risks of allele loss in small populations as a result of genetic drift (Barrett & Kohn, 1991; Elistrand & Elam, 1993). Alternatively the present results may reflect small sample sizes, though in the smallest extant populations even they represent a high proportion of the whole population and no sampling effect was detected. Variation in heterozygosity between the two smallest populations indicates that this method of assessing variability may not produce the same result as measuring allele diversity. This phenomenon has been noted by several authors (e.g. Nei, 1975; Barrett & Kohn, 1991) and an increase in heterozygosity is said to reflect rapid population growth following passage through a bottleneck. If this is the case in Lobelia urens, it suggests that the present pattern of genetic variation may be partly explained by differential passage through population bottlenecks. Because the biggest populations are those which have undergone the most extensive disturbance in recent years, the seed banks in these sites may have been sampled more effectively than in the smaller populations. Where no disturbance has occurred in recent years (Kilmington and Hinton Admiral), allele number has fallen. The small sample number and the wide range of significance values for Fis make it difficult to draw firm conclusions from the Kilmington data. The apparently high level of inbreeding suggested by low heterozygosity values at Hinton Admiral may also be a product of the lack of site management, and the consequent failure of release of seed from the seedbank. Because germination is inhibited in the dark, only those seeds on the surface can give rise to adult plants, and buried ones remain

21

dormant. Hence, recent recruitment has been from a limited number of parent plants and, because of this, the chance of mating among close relatives is increased. If this is accompanied by a degree of self-fertilisation (as suggested by seed set on isolated plants), a cycle of increased inbreeding is likely to be established. In order to test this hypothesis, it would be necessary to compare the population genetic structure of existing plants on managed and unmanaged sites with that of their respective seed banks. In conservation terms, the implication of the above suggestion is that the growing plants may only represent a proportion of the available genetic variation present at the site. Much of the potential variation may be locked into the seed bank and, in the absence of site management, will fail to contribute to future generations. It also means that population bottlenecks and genetic bottlenecks may not be the same, as the reserve of variation found in the seed bank will remain fairly constant in the short to medium term despite changes in numbers of growing plants. In the longer term this variation will gradually decrease as the seed bank becomes depleted through seed predation or reduced viability of ageing seed. Only when the site is disturbed will this trend be reversed. In summary, each population of Lobelia urens should be regarded as an important contributor to the overall amount of genetic variation present and all populations should, therefore, be conserved. In order to maintain the maximum amount of variation within each population, it should be managed by periodic site disturbance, as the number of plants begins to decline, to allow germination from other genotypes. Similar conservation management prescriptions may need to be applied to other species in which inbreeding and banking of seed are significant features of the reproductive biology. ACKNOWLEDGEMENTS The authors are grateful to the owners and managers of all the sites for allowing us to collect material from the plants for this study. Special thanks to Janet Dinsdale, University of Plymouth, and Chris Gliddon, University College Bangor, for their valuable comments and discussion.

REFERENCES

Barrett, S. C. H. & Kohn, J. R. (1991). Genetic and evolutionary consequences of small population size in plants: implications for conservation. In Genetics and conservation o f rare plants, ed. D. A. Falk & K. E. Holsinger. Oxford University Press, New York, Oxford, pp. 3-30. Bates, A. (1992). Population surveys and habitat management of Lobelia urens. Recording Dorset, 2, 17-20. Brightmore, D. (1968). Biological flora of the British Isles: Lobelia urens L. J. Ecol., 56, 613~20. Clarke, W. A. (1778). First records of botanical flowering plants - - Lobelia urens L. J. Bot. Lond., 31,279.

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