| Summary: | The Lincolnshire Limewoods are a group of Ancient Semi Natural Woodlands within Central Lincolnshire that include the nationally important Bardney Limewoods National Nature Reserve. The woods, although fragmented and isolated by tracts of agricultural land, are reservoirs of biodiversity and contain large populations of Tilia cordata Mill. The current management aims are to increase the biodiversity within the woods, as well as to extend and improve the connectivity between the woodlands, with new planting. An understanding of the genetic diversity and structure of the Limewoods, both as individual woods and by comparison with woods from other regions of Britain, will help to inform management decisions. A pilot study was undertaken using RAPD markers which demonstrated the potential for these markers to amplify and identify individual T. cordata trees. Dominant markers are less informative than co-dominant markers, especially when trees may be closely related, and to facilitate this study a T. cordata enriched microsatellite library was constructed. The ten microsatellite loci designed for the genetic study amplified both T. cordata and closely related Tilia platyphyllos Scop. and were also able to identify hybridisation between the two species. T. platyphyllos and hybrid trees were detected in eleven of the Lincolnshire Limewoods and were associated with identification of private alleles within the T. cordata populations. The high levels of genetic diversity and low genetic variance which were found show that the Lincolnshire Limewoods’ populations are all similar. Comparison with populations from outside Lincolnshire show similar genetic diversity, with AMOVA ii conducted over all populations showing that only 4% of the variation could be allocated between the populations whereas 82% was allocated between the individuals within the populations. Weak isolation by distance was identified and would suggest that the Lincolnshire Limewoods should be treated as a single population group for management purposes. To increase the likelihood that T. cordata is used for replanting schemes, seeds should be taken from woods without hybrid populations and, preferably, should be screened to identify and exclude F1 hybrids. Tissue culture was considered as a potential source of planting stock for the Lincolnshire Limewoods to provide trees of known provenance with potentially rejuvenated characteristics. Preliminary tissue culture investigation showed that the initial treatment and age of the tissue before sterilisation is important in controlling contamination after sterilisation. Tissue collected in the spring, prior to leaf emergence, with the buds allowed to develop under clean laboratory conditions resulted in a reduction in the number of contaminated explants. In explant culture, roots were induced with application of the auxin naphthaleneacetic acid (NAA) and microshoots were induced with a combination of NAA and cytokinin, 6-benzyl-aminopurine (BAP). However, no rooted plantlets were produced. Further investigation into the use of somatic embryos as an explant source should now be considered as a possible way of reducing the chronic contamination that was experienced using axial buds.
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