PAST, PRESENT AND FUTURE? CONSERVATION PRACTICE AND THE CONSERVATION GENETICS OF PLANT POPULATIONS

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PAST, PRESENT ... AND FUTURE? CONSERVATION PRACTICE AND THE CONSERVATION GENETICS OF PLANT POPULATIONS. Q.O.N. KAY As new, effective and rapid molecular techniques for the determination of patterns of genetic variation have been introduced during the past 20-25 years, the remarkable diversity of genetic situations in plant populations, both within and between species, has become increasingly clear. The real situations, revealed by isozyme and DNA analysis, often prove to differ from those that would in the past have been predicted from knowledge of the apparent breeding system and population structure of the species. Such divergence from classical expectation appears to be particularly frequent in rare and declining species, which are usually those of greatest immediate conservation concern.

Knowledge of these genetic structures and patterns of genetic variation is centrally important for plant conservation.

Sources of genetic data What sources of genetic data are available for conservation programmes?

They are:Classical, primarily morphological taxonomic data.

Although taxonomic distinctions are usually based on genetic differences, these differences may sometimes be extremely small and the distinctions dependent upon subjective judgement or opinion. In many cases, however, classical taxonomic judgements are a good guide to genetic relationships; fortunately so, because these judgements are the normal basis on which species, subspecies etc. are recognised for conservation purposes.

Classical studies of ecotypic variation, from Turesson (or earlier) onwards.

These are usually based on the demonstration of genetically based variation by comparative cultivation, for example in Succisa pratensis (Devil’s-bit Scabious). When available, these studies are extremely useful as indications of the pattern and degree of geographic and ecotypic genetic variation in a species. This variation is usually adaptive and thus subject to natural selection.

Classical genecological studies of visible or easily detected polymorphisms For example leaf-pattern, flower colour, cyanogenesis, perhaps also metal tolerance and self-incompatibility alleles. These studies are indicative of breeding systems and likely levels of variation, and sometimes significant for conservation purposes (SI allele polymorphisms especially so) but in general are of limited scope, and only available for a few species (e.g. Trifolium repens (White Clover) , Lotus corniculatus (Common Bird’s-foot Trefoil) and Papaver rhoeas (Common Poppy)).

Studies of molecular variation and polymorphism; isozyme analysis, various DNA techniques from RAPD analysis to direct DNA sequencing.

These new techniques have proved to be enormously valuable and informative, although each technique has its strengths and weaknesses. For use in conservation work, a technique should ideally be fast and cheap, widely

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applicable, maximally and relevantly informative, reliably repeatable, and requiring minimal amounts of plant material. For plant conservation work, the best techniques that are currently available are: 1. Isozyme analysis. This technique, developed during the 1970s but subsequently much refined and improved, is fast, relatively cheap, uses only small amounts of plant material (a postage-stamp sized part of a leaf), has wide applicability and is repeatable and extremely informative (Hamrick & Godt, 1989; Vekemans & Jacquemart, 1997). A practical limitation of the technique is that the extraction and electrophoretic separation of active enzymes, its essential feature, is easy in some plant groups and species, using standard procedures, but for various possible reasons difficult or impossible in other groups and species, sometimes unpredictably so. Trials of alternative procedures may or may not result in success and are likely to be time-consuming. A particular strength of the technique is that by virtue of its enzyme morph comparison it compares single-copy coding DNA sequences that are expressed phenotypically, rather than the random assortment of DNA sequences, often non-coding and highly repetitive, that are compared by the direct DNA comparison techniques. This can, however, also be a limitation on its sensitivity because these single-copy coding sequences of DNA are tbe most conserved regions of DNA, so diverge more slowly than other region.

2. RAPD (Random Amplified Polymorphic DNA) analysis. Like a number of related techniques this uses PCR (polymerase chain reaction) amplification, so that quite small amounts of plant material, comparable to those required for isozyme analysis, are required to provide the initial DNA samples. Technically it is somewhat more expensive and demanding than isozyme analysis, but it is similarly rapid and widely applicable, and has been used in many studies, often in conjunction with isozyme analysis, usually providing similar results, potentially more informative in some ways (greater resolution) but more limited in others. In comparison with isozyme analysis, its drawbacks include its lower reliability and reproducibility, the possibility of amplification of DNA from the wrong organism, and its dependence on anonymous genomic sequences). A particular advantage is that RAPD analysis may be practicable for plants in which isozyme analysis fails.

Many other techniques have been developed (for example RFLP, restriction fragment length polymorphism analysis), some more sensitive than isozyme or RAPD analysis, but often requiring substantially greater quantities of plant material and/or lengthy and relatively costly procedures and thus less suitable or impracticable for the population analyses involved in conservation work.

These techniques, up to and including complete DNA sequencing, have often been used in phylogenetic studies. Those involving comparisons of chloroplast DNA (cpDNA) and mitochondrial DNA (mtDNA) are becoming increasingly relevant to population studies, especially in phylogeographic studies of the origin, history and migration routes of populations (e.g. Quercus spp., oaks, Ferris et al., 1995, 1998).

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Why is genetic data important in conservation? What can it tell us?

Genetic data is of great and central importance in plant conservation work. It provides essential information on a series of basic questions that must be answered if a conservation programme is to be appropriately targeted and to have a real prospect of long-term success. Examples of these basic points are: 1. The distinctness and relationships of the population, populations or taxon that we are concerned with. Examples from recent molecular work include Ulmus plotii (Plot’s Elm) (shown to be genetically distinct but apparently a single clone, Coleman, Hollingsworth & Hollingsworth, 2000), Sorbus (Whitebeam) microspecies (shown to be clearly distinct units in some cases, e.g. S. minima, S. leyana, but composite groups of genetically distinct and sometimes unrelated genotypes in other cases, e.g. S. porrigentiformis, Lemche, 1999) and the investigation of the newly described orchid species Epipactis youngiana (Young’s Helleborine) by Harris & Abbott (1997) which showed that it was not clearly distinct from the common and genetically variable E. helleborine (Broad-leaved Helleborine).

2. The level and patterns of genetic variation within the population, population-group or species. An example is the study of Scottish populations of the rare arctic-alpine saxifrage Saxifraga rivularis (Highland Saxifrage), in which isozyme and RAPD analysis (Hollingsworth et al., 1998) has shown that only five molecular phenotypes occur in the eleven widely separated populations that were studied, and that all but one (Lochnagar) of the populations were monomorphic.

3. In species with clonal spread, the true number of genetic individuals (genets) in a population - one clone, a few clones, or many clones?

Examples from recent work on common and widespread species range from Anemone nemorosa (Wood Anemone) in which despite its apparently vigorous clonal spread and the apparent likelihood of single- or few-clone population structures recent work has shown that populations in reality consist of large numbers of genetically different genets (Holderegger, Stehlik & Schneller, 1998; Stehlik & Holderegger, 2000) to Fallopia japonica (Japanese Knotweed) in which all British populations are a single uniform clone (Hollingsworth & Bailey, 2000). More complex situations are found in rare and local species, for example the rare dioecious Georgia shrub Lindera melissifolia (Pondberry) in which most populations have been shown to consist of one or a few clones (Godt & Hamrick, 1996).

4. Levels of outbreeding (heterozygosity). In polymorphic populations of a diploid species, or polyploids in which true heterozygotes can be distinguished from ‘fixed’ heterozygotes, isozyme analysis provides a particularly sensitive and accurate measure of levels of outbreeding and gene-flow. Among many examples, British populations of Mibora minima (Early Sand-grass), a diminutive diploid annual grass which classical theory would have predicted to be inbreeding and largely or entirely homozygous, were shown by isozyme analysis to be highly heterozygous and largely outbreeding (John, 1992; Kay & John, 1997).

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5. The possible or probable history of a population or group of populations for example the sites of Pleistocene refugia, migration routes and relationships to other populations (phylogeography) and also the possible occurrence of bottlenecks of low population size and genetic drift. Isozyme analysis and cpDNA have been particularly useful in phylogeographic studies, for example those of Viola rupestris (Teesdale Violet) in Scandinavia (isozyme analysis, Nordal & Jonsell, 1998) and Quercus spp. (oaks) in Europe (cpDNA, Ferris et al., 1995, 1998.)

6. Potential genetic responses (adaptation) to future changes in environmental pressures or population size. Here molecular studies are often the only possible source of essential data on overall levels of genetic variation, differentiation between populations, and actual and potential gene-flow, including hybridization and introgression.

7. The possible effects of different conservation measures, and the conservation measures that are likely to be most appropriate for a particular population, population group or species. This centrally important point is discussed in the next section.

Without genetic data, what damage may be done by re-introduction and other active conservation measures?

What are the potential dangers of conservation programmes - however wellintentioned - in which genetic data about the populations that are involved are lacking, or unknown to those involved?

1. Loss of the historical record. The genetic composition of the natural population may have documented its history, for example by the presence or absence of particular isozyme alleles or cpDNA types. Introduction of alien genotypes is likely to destroy or obscure this record of thousands of years of history. Rare and local species with highly disjunct populations are at particular risk. This potential loss is an especially distressing and irreversible danger.

2. Direct (first-generation) loss of adaptedness. In rare, local or ecologically specialised species, the existing population, whether or not it now appears to be inbred and relatively lacking in genetic variation, has in all probability survived at its present site, or at similar sites in the same metapopulation area, for thousands of years. This survival is the strongest possible evidence of its genetic adaption to local conditions. The introduction of alien, unadapted genotypes may lead to complete loss of this adaptedness, or to the production of unsustainable levels of maladapted offspring. The same considerations apply to locally adapted populations of ecologically and/or geographically wide-ranging species, for example Crataegus monogyna (Hawthorn) and Succisa pratensis.

3. Secondary (F2 onwards) loss of adaptedness through breakdown of coadapted gene complexes (epistasis). A typical pattern here is for the F1 generation produced by hybridisation between the native and alien genotypes to be more vigorous and successful than either, but for the F2s

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to be inferior, with F3 and later generations continuing the decline. It is caused by the recombination and consequent breakdown of disparate coadapted multilocus gene complexes involved in the adaptation of the native and alien genotypes to their particular local conditions. Far from being an esoteric genetic effect, it has recently been demonstrated by Keller, Kollmann and Edwards (2000) to be a general effect for Agrostemma githago (Corncockle) and Papaver rhoeas (Common Poppy) from a variety of origins; they consider that it is likely to be a widespread consequence of the use of ‘wildlife strip’ wildflower mixture consisting of seeds of alien origin, contributing to the loss or decline of surviving native or long-established local populations of the species included in these mixtures.

4. Introduced alien genotypes may be, initially at least, more successful than the original native population, reproductively or competitively, or simply through pressure of numbers (minority type disadvantage affecting a numerically inferior native population), with the result that the native genotypes are swamped or lost (Keller, Kollmann & Edwards, 2000).

Thus, instead of conserving the native population, one has destroyed it, despite superficial appearances to the contrary. In the long term, the introduced genotypes may not survive, lacking the adaptations to longterm survival through environmental fluctuations that were possessed by the original native population; on the other hand, they may be equally successful, more successful, or even undesirably invasive (see next section).

5. In some cases introduced alien genotypes may become, or already be, invasive, with a wider ecological range that the original native populations, perhaps invading new sites and habitats and causing unforeseen ecological changes. Possible examples in the British Isles include Chamerion angustifolium (Rosebay Willowherb) and tetraploid Leucanthemum vulgare (Ox-eye Daisy).

6. Unforeseen interactions with diseases and herbivores may have severe effects. These may have both genetic (differential sensitivity of native and introduced types) and non-genetic (introduction of diseases and herbivores with introduced plants) components. At one extreme, the vegetative vigour of an introduced genotype, giving it initial and perhaps decisive competitive superiority over the native genotype, may be due to its lack of resource-demanding inbuilt resistance to a disease or herbivore which is perhaps temporarily - not present at the time of introduction. Lack of thorns in Crataegus monogyna (Jones, Hayes & Sackville-Hamilton, in press 2001) is one such example. At the other extreme, the introduced genotype, but not the native genotype, may be resistant to a disease or herbivore which is carried by the introduced plants and introduced with them.

7. During conservation programmes (for example during routine monitoring) it is common for visits to be made successively to populations which are

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Suffolk Natural History, Vol. 37 isolated from one another and not otherwise in contact. Such visits may unintentionally result in the transfer of seeds or other propagules, or of specific diseases or herbivores, between populations, for example on the wheels of vehicles (the Land-Rover effect) or on clothing or boots.

Although seed transfer might be beneficial in some cases, it is in general undesirable, especially so when genetic data is not available; transfer of specific deleterious diseases and herbivores is obviously undesirable at any time. Appropriate precautions against unintentional transfers should therefore be taken.

What should be done to minimise or eliminate these and other potentially harmful consequences of conservation, re-introduction and translocation programmes?

One possibility is to regard introductions, re-introductions and translocations as unacceptable and to rule them out as permissible parts of conservation programmes. A strong case can be made out for this view, and in general they should probably be regarded as a last resort, habitat protection and maintenance being the preferred primary approach. Nevertheless, in the present situation of drastic, largely man-induced habitat destruction, fragmentation and change, with, possibly, the superimposed effects of climatic change, active conservation measures involving plant propagation and transfer, both in situ and ex situ, including introductions, re-introductions and translocations, are certainly justified, at least in particular cases (e.g. Falk, Millar & Olwell, 1996). They should however not be treated as an expected component of any conservation programme, and if they are included it is essential that they should be carefully planned to minimise or eliminate possible harmful effects. The hazards of rare plant reintroduction and recovery programmes, and guidelines and precautions for conducting them, were discussed in detail by Falk, Millar and Olwell (1996) and, in a British context,

by Kay and John (1994), Bullock et al. (1996) and Fleming and Sydes (1997).

What precautions should be taken to minimise or eliminate possible harmful effects of introduction, re-introduction or translocation programmes?

A wide range of suggested guidelines and policy statements, varying in their applicability and rigour, have been produced by conservation organisations concerned with various types of translocation and introduction or reintroduction of animals and plants in the UK and elsewhere (reviewed by Bullock et al. 1996). The following broad guidelines apply to plants and highlight some points where particular caution is advisable.

If the patterns of genetic variation in the population, population group or species are not known, seeds or other propagules should be collected only from populations in the immediate locality. Definitions of this will vary from species to species and situation to situation; a radius of about 10 to 20 km seems to be an appropriate target in Britain, although greater distances may obviously have to be involved sometimes. Sampling procedures should be appropriate to the particular case, and carefully planned and recorded. The source population should grow in a similar habitat. If harvesting techniques

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are used for a multi-species introduction the source community should not include invasive or alien species or genotypes. A species should not be introduced to a site where it has occurred in the past unless it is absolutely certain that it is extinct there. All these considerations apply across the board, from highly endangered rare species like Rumex rupestris (Shore Dock) (Kay, 1996) to the currently or formerly common species like Agrostemma githago, Papaver rhoeas (Keller, Kollmann & Edwards, 2000), Chrysanthemum segetum (Corn Marigold), Leucanthemum vulgare (Ox-eye Daisy) and Lotus corniculatus (Bird’s-foot Trefoil) that are included in commercially available ‘wildflower’ and ‘meadow’ mixtures.

The new national code of practice for the collection and sale of native wildflower seeds and propagules introduced in August 2000 by Plantlife and Flora Locale (Anon., 2000) incorporates guidelines that broadly incorporate most of these principles, and enable the precise origin of material on sale to be determined, but do not define the size of a ‘local area’. The Weald Meadows Initiative (Brickwood, Hobbs & Davis, 2000) provides a good example of the application of these principles within a known local area.

If the pattern of genetic variation is known, collection from more distant sites may be permissible. Possible examples here include known apomicts (e.g. Hieracium sect. Alpina, Shi et al., 1996) and vegetatively propagated uniclonal taxa like Ulmus plotii (Coleman, Hollingsworth & Hollingsworth, 2000). In general, however, the precautionary rule of collecting only from populations growing in similar habitats in the immediate locality should still apply.

Ex situ cultivation of material that is later used for introduction, reintroduction or translocation should be avoided, or used only with extreme precautions against its many potential dangers. These dangers include genetic change (genetic drift and/or adaptation to artificial conditions with consequent loss of adaptation to the natural environment, or hybridisation between plants of different origin with the same possible result), and at the non-genetic level the acquisition of pests and diseases present in the garden environment, and contamination with seeds or propagules of other species, particularly in accompanying soil.

Important genetic guidelines (Fleming & Sydes, 1997) are that plants being grown for return to the wild should be derived directly from wild propagules wherever possible, and that if material needs to be bulked up the number of sexual generations should be strictly limited. For longer-term ex situ maintenance, which is certainly necessary for the most endangered populations, perennial species should be maintained without sexual regeneration unless they are apomictic or strictly selfing, while outcrossing annuals and short-lived or semelparous perennials are probably best maintained in seed banks. This ex situ material will function both as an ultimate reserve and as a source of material for research. Ideally, cultivated population samples should be maintained at least in duplicate, at separate sites, and seed banks, which are compact and efficient but dangerously vulnerable to political change and other factors which may prevent their continued existence or satisfactory upkeep, should also be maintained at least in duplicate, preferably at geographically and politically separate sites.

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What on-site (in situ) conservation actions are most likely to aid the longterm survival and increase the size of endangered populations?

Protection of the habitat and maintenance of suitable habitat conditions are, of course, obvious and basic components of any conservation programme.

Beyond this, there are a number of simple, effective and (in nearly all cases) genetically benign actions that can be taken to aid long-term survival. These include: Assisted pollination within populations. Sparse populations frequently suffer from inadequate pollination by natural vectors, resulting in poor seed production and limited gene-flow within the population. Knowledge of the genotypes of individual plants within the population may increase the effectiveness of this measure as a means of maintaining levels of genetic variation.

Assisted dispersal and placement of seeds and propagules within the population. Bias towards the most prolific seeders within the population should be avoided (Fleming & Sydes, 1997).

Continuing genetic monitoring, as well as other forms of monitoring of the population.

Provision of additional suitable habitat.

Provision of escape routes and migration corridors to other actual or potential areas of suitable habitat. At the local level, this is particularly important for species of seral or temporary habitats which cannot easily be maintained in situ (e.g. newly-formed dune-slack specialists like Gentianella uliginosa (Dune Gentian) and Liparis loeselii (Fen Orchid)). Over a longer time-scale and more generally, it may sometimes be possible to provide local escape routes for populations that are likely to be affected by global climatic change, for example to higher altitudes or cooler aspects for species affected by global warming, and to drier sites for species affected by increasing rainfall.

What needs to be done?

What needs to be done to prevent unintentional future harm to plant and animal populations resulting from well-meaning but misdirected conservation actions, and to remedy or minimise damage that might already have been done?

First, UK biodiversity action programmes need to be provided with a firmer basis in terms of knowledge of existing genetic information, the availability of genetic survey and monitoring capability, and general understanding of the central importance of genetic factors. It must be realised that it is essential to take steps to determine the actual genetic structures and patterns of genetic variation in the species and populations that are included in the action programmes, and this requirement should be written into these programmes as a matter of course.

Awareness of existing genetic information needs to be improved. Although at present it is probably true to say that relatively few conservation-orientated or phylogeographic molecular genetic studies have been made in Britain, in

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comparison with the much greater numbers of such studies that have been made in the United States and mainland Europe, much information relevant to the British flora does exist both from studies made here and from studies of overseas (mainly continental European and Mediterranean) populations of species that also occur in Britain. Examples of molecular genetic studies made in Britain and Ireland include the studies of British populations of some tree species by Richard Ennos, Colin Ferris and others, of Potamogeton (pondweeds) and several other species by Peter Hollingsworth and coworkers, of Senecio vulgaris and related species in many papers by Richard Abbott’s group, of Polygala species (milkworts) by Andrew Lack (Lack & Kay, 1987, 1988), of several species of grasslands and open habitats in a wideranging survey for CCW (Kay & John, 1994, 1995), and of a few arctic and alpine species, including Lloydia serotina (Jones & Gliddon, 1999), Primula scotica (Glover & Abbott, 1995) and Saxifraga rivularis (Hollingsworth et al., 1998). A comprehensive review and bibliography of molecular genetic studies of intraspecific variation in species that occur in the British flora is needed, together with a regularly updated guide to work in progress and recently published, similar to the guide to experimental taxonomic work relevant to the British flora that was compiled by D.H. Valentine and others in the 1960s and 1970s.

Provision of genetic survey and monitoring capacity requires central funding, but would represent a very minor part of total expenditure on biodiversity action. Establishment or expansion of units attached to the conservation agencies (English Nature and its counterparts in Wales, Scotland and Ireland) and to the national Botanic Gardens (including those in Wales and Ireland), together with more flexible and conservation-oriented funding of work done in universities and research stations, is needed. Within a context of central Government support and understanding similar to that found in the United States and continental European countries, great potential benefits would come from a flexible and properly financed programme of this type, integrated with the overall biodiversity action initiative.

Finally, genetic assessments of existing conservation programmes need to be made, where they do not already exist. These assessments should indicate the relevant actions that need to be taken (e.g. genetic survey and monitoring) and the precautions, guidance and possible remedial measures that should be incorporated in the programmes. They should cover schemes affecting common or formerly common species (e.g. arable wildflower strips, wildflower meadow creation) as well as the action plans for rare and declining species.

References Anon. (2000). Planting with wildlife in mind: Code of Practice for collectors, growers and suppliers of native flora. Flora Locale, Newbury, England.

Brickwood, D. Hobbs, R. & Davis, R. (2000). Weald Meadows leads way on new ‘Native Code’. Biodiversity News 13: 5.

Bullock, J. M., Hodder, K. H., Manchester, S. J. & Stevenson, M. J. (1996).

Review of information, policy and legislation on species translocation.

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Coleman, M., Hollingsworth, M. & Hollingsworth, P. M. (2000). Application of RAPDs to the critical taxonomy of the English endemic elm Ulmus plotii Druce. Botanical Journal of the Linnean Society 133: 241–262.

Falk, D. A., Millar, C. I. & Olwell, M. (1996). Restoring diversity: strategies for reintroduction of endangered plants. Island Press, Washington.

Ferris, C., King, R. A., Vainola, R. & Hewitt, G. M. (1998). Chloroplast DNA recognizes three refugial sources of European oaks and suggests independent eastern and western immigrations to Finland. Heredity 80 PN 5: 584–593.

Ferris, C., Oliver, R. P., Davy, A. J. & Hewitt, G. M. (1995). Using chloroplast DNA to trace postglacial migration routes of oaks into Britain.

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Glover, B. J. & Abbott, R. J. (1995). Low genetic diversity in the Scottish endemic Primula scotica Hook. New Phytologist 129: 147–153.

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Hamrick, J. L. & Godt, M. J. (1989). Allozyme diversity in plant species, in Brown, A. H., Clegg, M. T., Kahler, A. L. & Weir, B. S., eds., Population genetics and germplasm resources in crop improvement, pp. 44–64.

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Harris, S. A. & Abbott, R. J. (1997). Isozyme analysis of the reported origin of a new hybrid orchid species, Epipactis youngiana (Young’s helleborine), in the British Isles. Heredity 79: 402–407.

Holderegger, R., Stehlik, I. & Schneller, J. J. (1998). Estimation of the relative importance of sexual and vegetative reproduction in the clonal woodland herb Anemone nemorosa. Oecologia 117: 105–107.

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Hollingsworth, P. M., Tebbitt, M., Watson, K. J. & Gornall, R. J. (1998).

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Jones, A. T., Hayes, M. J. & Sackville Hamilton, N.R. (in press, 2001). The effect of provenance on the performance of Crataegus monogyna in hedges. Journal of Applied Ecology 38.

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Kay, Q. O. N. & John, R.F. (1994). Population genetics and demographic ecology of some scarce and declining vascular plants of Welsh lowland grassland and related habitats. Science Report no. 93, Countryside Council for Wales, Bangor, Wales.

Kay, Q. O. N. & John, R. F. (1995). The conservation of scarce and declining plant species in lowland Wales: population genetics, demographic ecology and recommendations for future conservation in 32 species of lowland grassland and related habitats. Science Report no. 110, Countryside Council for Wales, Bangor, Wales.

Kay, Q. O. N. & John, R. F.(1997). Patterns of variation in relation to the conservation of rare and declining plant species, in Tew, T., Crawford, T., Spencer, J., Stevens, D., Usher, M. & Warren, J., eds., The role of genetics in conserving small populations, pp. 41–55. JNCC, Peterborough.

Keller, M., Kollmann, J. & Edwards, P. J. (2000). Genetic introgression from distant provenances reduces fitness in local weed populations. Journal of Applied Ecology 37: 647–659.

Lack, A. J. & Kay, Q. O. N. (1987). Genetic structure, gene flow and reproductive ecology in sand-dune populations of Polygala vulgaris.

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Lack, A. J. & Kay, Q. O. N. (1988). Allele frequencies, genetic relationships and heterozygosity in Polygala vulgaris populations from contrasting habitats in southern Britain. Biological Journal of the Linnean Society 34: 119–147.

Lemche, E. B. (1999). The origins and interactions of British Sorbus species.

PhD. Thesis, University of Cambridge.

Nordal, I. & Jonsell, B. (1998). A phylogeographic analysis of Viola rupestris: three post- glacial immigration routes into the Nordic area? Botanical Journal of the Linnean Society 128: 105–122.

Shi, Y., Gornall, R. J., Draper, J. & Stace, C. A. (1996). Intraspecific molecular variation in Hieracium sect. Alpina (Asteraceae), an apomictic group. Folia Geobotanica & Phytotaxonomica 31: 305–313.

Stehlik, I. & Holderegger, R. (2000). Spatial genetic structure and clonal diversity of Anemone nemorosa in late successional deciduous woodlands of Central Europe. Journal of Ecology 88: 424–435.

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Quentin Kay School of Biological Sciences University of Wales Swansea SA2 8PP

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