Evaluation of Introduced and Naturalised Populations of Red Clover(<Emphasis Type="Italic">Trifolium pratense</Emphasis> L.) at Pergamino EEA-INTA,Argentina

Red clover (Trifolium pratense L.) is a temperate forage species that is extensively used in Argentina, but little effort in breeding has been made so far. Meanwhile, red clover breeding is strong in Europe and North America. In order to contribute to the utilisation of red clover genetic resources, 39 accessions of different origins were evaluated at Pergamino Inta Experimental Station. A broad phenotypic variation was detected for many of the 14 attributes considered. Variables that discriminate populations are related to seed production and winter yield. Two different groups clustered according to morphologic and productive mean attributes, and these groups were related to geographic origin of accessions.     

Microsatellite variability within and among local landrace populations of tea, Camellia sinensis (L.) O. Kuntze, in Kyoto, Japan

Genetic variability within and among eight landrace populations of tea (Camellia sinensis (L.) O. Kuntze) located in southern Kyoto, Japan, was surveyed with six microsatellite markers. The average number of alleles per locus was 3.83 to 4.67 for landrace populations, whereas the corresponding value among modern cultivars and breeding lines was 6.63. Expected heterozygosity values averaged over loci within landrace populations ranged from 0.498 to 0.723. A similar level of variation, 0.682, was observed for cultivars and breeding lines. High fixation index values (0.177–0.417) for each population are consistent with biparental inbreeding within the population. Genetic differentiation among local populations was extremely low with F _ST = 0.062, although AMOVA revealed significant differentiation among landrace populations. We propose that these populations share a common ancestral gene pool and that some degree of artificial selection within each population has been performed by local farmers. Neighbor-joining analysis revealed that genetic relationships among populations reflect geographical location of populations. This might result from more frequent genetic exchange by nearby farmers.     

RAPD and AFLP assessment of genetic variation in a landrace of pepper (Capsicum annuum L.), grown in North-West Italy

In several regions of Italy as well as other parts of southern Europe, the heterogeneity of the land, the climate and the soil favour the survival in cultivation of a large number of landraces specifically adapted to local conditions. Knowledge on the level and distribution of their genetic variation can help to develop appropriate strategies, in order to suistainably manage in situ these germplasm resources at risk of genetic erosion. C. annuum is an herbaceous diploid species and is considered to be self-pollinating, although different rates of out-crossing have been recorded. We used random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) markers to assess genetic diversity within and between five populations of a landrace of Capsicum annuum L., grown in a limited area in north-west Italy and locally known as Cuneo pepper. Partitioning the genetic variation with Shannon's diversity index revealed that 41.6% occurred between and 58.4% within populations. Analogous results were obtained when the analysis was based only on RAPD or AFLP markers. However, AFLP was more reliable, since a lower range of variation was observed among primer combinations in detecting the two components of genetic variation. Notwithstanding the rather high level of within genetic variation detected, the five populations were clearly differentiated and differed in the frequency of alleles exclusive and/or present at very low frequencies. Our results show the need for accurate estimation of allele frequencies, in order to identify populations to which priority should be given for dynamic conservation of landraces.     

Quantitative and Qualitative Traits of Natural Ecotypes of Perennial Grasses (Dactylis glomerata L., Festuca arundinacea Schreb., Phalaris tuberosa L., Brachypodium rupestre (Host) R. et S.) Collected in Southern Italy

In 1999, natural populations of Dactylis glomerata L., Festuca arundinacea Schreb., Phalaris tuberosa L. and the macrotherm species Brachypodium rupestre (Host) R. et S., were collected as seed in Southern Italy (Apulia, Basilicata and Campania regions) to evaluate the potential as forage of native germplasm under dry conditions, using available commercial cultivars as controls. The persistence of accessions, biometric, phenologic, productive and qualitative parameters of forage have been studied for a 3-year period (2000–2002) in a typical Mediterranean environment. D. glomerata is widely present in the studied area with two subspecies (subsp. glomerata L., subsp. hispanica (Roth) Nyman). The research pointed out significant differences in plant size, earliness and single plant dry matter (DM) production. All the natural ecotypes belonging to this species showed higher persistence than the control cultivars. Some natural ecotypes appeared to have potential for improving DM yield (subsp. glomerata ecotypes) and reducing neutral detergent fibre (NDF) content (subsp. hispanica ecotypes). Natural ecotypes belonging to F. arundinacea, P. tuberosa, and B. rupestre are sporadically present in the studied area. Among them, two ecotypes of P. tuberosa, showing higher winter growth and earliness compared to cv. ‘Holdfast’, seem to be interesting for a future breeding programme.     

Genetic relationships and diversity among Brazilian cultivars and landraces of common beans (Phaseolus vulgaris L.) revealed by AFLP markers

The genetic variation and relationships among 31 accessions of Phaseolus vulgaris L., and two representatives of Vigna unguiculata L., were evaluated by AFLP analysis. A total of 263 DNA fragments across all materials were scored using nine primer combinations, averaging 32 per primer. More than 95% of the amplification products showed polymorphism, indicating high variation at the DNA level among these accessions. Pair-wise genetic similarity (Jaccard's coefficient) ranged from 0.553 to 0.840, with a mean of 0.765. Twenty-three accessions (70%) clustered into three groups. A majority of the commercial cultivars (91%) clustered within a single group, whereas the landraces were distributed along all the variation. An apparent correlation with phaseolin types was detected. Results of this study suggest that Brazilian landraces truly represent the overall genetic variability of Phaseolus vulgaris, confirming the multiple origins of these materials, and their potential as a source of variation for breeding programs.     

Genetic Effects on Grain Characteristics of <Emphasis Type="Italic">indica</Emphasis> Black Rice and their Useson Indirect Selections for some Mineral Element Contents in Grains

Complete diallel crosses with Oryza sativa L., six varieties of black rice and one variety of aromatic white rice were conducted to study the seed, maternal and cytoplasmic genetic effects on grain characteristics such as 100-grain weight, length, width and shape and their genetic correlations with Fe, Zn, Mn and P mineral contents in kernels by using the genetic model on quantitative traits of seed in parents and their F₁s and F₂s. The seed genetic effects were found more important than the maternal genetic effects for grain characteristics, and seed additive effects constituted a major part of their genetic effects. The heritabilities of seed effects were high for 100-grain weight, width and shape and moderate for grain length. Single seed selection based on the 100-grain weight, width and shape was advocated in early generations, whereas single plant and seed selection based on grain length were advocated in late generations. Significant genetic correlations including seed additive, dominance, cytoplasmic, maternal additive and dominance between 100-grain weight, length, width and shape and Fe, Zn, Mn and P mineral contents were observed. Indirect selection of grain characteristics may be one of the breeding methods to select for higher contents of Fe, Zn, Mn and P in black pericarp indica rice.     

Germplasm Sources for Improvement of Forage Quality in Lotus corniculatus L. and L. uliginosus Schkuhr (Fabaceae)

Extensive germplasm resources are present in collections of Lotus corniculatus and L. uliginosus, two forage species that are valuable in situations where other legumes are not persistent. Naturalised populations in many regions are the primary gene pool for genetic improvement of these species and interspecific hybrids are an important source of variation for agronomic and forage quality traits. Although previous research has provided evidence that selection for optimal levels of condensed tannins and herbage productivity are feasible breeding objectives, less is known about correlations of condensed tannin with other parameters of nutritive value. A germplasm collection of 38 accessions in both species from 9 geographic regions was used as a data base to determine these correlations. Forage of each accession was sampled in two successive years from a field site in south-eastern Australia and analysed for condensed tannin, in vitro digestible dry matter, nitrogen, acid detergent fibre and neutral detergent fibre. In both species condensed tannin was negatively correlated with in vitro digestible dry matter and nitrogen. This result helped to identify potentially valuable accessions with low condensed tannin (<4% of dry weight) and high in vitro digestible dry matter (>70%). One of these was an interspecific hybrid of L. uliginosus and L. corniculatus, confirming the view that interspecific hybridization would be a valuable avenue for improvement of agronomic and forage quality characters in Lotus species.     

Population diversity of the Vicia sativa agg. (Fabaceae) in the flora of the former USSR deduced from RAPD and seed protein analyses

The genetic diversity of 58 wild and weedy populations representing taxa within the V. sativa aggregate from the former USSR, 4 cultivars of V. sativa, 2 accessions of V. cordata and 3 accessions of V. macrocarpa from Mediterranean countries were analysed using randomly amplified DNA fragments (RAPDs) and seed protein electrophoresis (SDS-PAGE). Interspecific variation between taxa in the V. sativa aggregate could readily be detected using both techniques. RAPDs and seed protein patterns were found to be an effective means of identifying accessions that cannot be identified clearly by morphological criteria alone. RAPD and seed protein analysis revealed a clear relationship between observed genetic variation of populations and their geographical distribution. Populations from each region had their own gene pools. Geographical variation was detected in V. segetalis. The degree of genetic divergence between local populations was usually related to proximity. In several locations where wild and weedy populations of different V. sativa agg. taxa grow sympatrically, intermediate forms could be detected at the DNA and protein levels. Both RAPD and seed protein analysis support the view that the V. sativa aggregate consists of 8 taxa warranting recognition at the species level. Several species in this aggregate are evolving intra-specific groups which can readily be detected at the molecular level.     

Extensive introgression of Middle American germplasm into Chilean common bean cultivars

Summary The genetic diversity of 95. representative Chilean common bean (Phaseolus vulgaris L.) landraces was analyzed using phaseolin seed protein and eight isozyme systems as genetic markers. Four types of phaseolin were found, “C”, “T”, “S” and “H”, in decreasing order of frequency. Each type had a different distribution between the Northern and Southern regions of the country. Nei’s genetic distance based on isozyme diversity indicated that a high percentage of the total variation found in this sample occurred between landraces and only a small percentage of the variation was detected within populations. Cluster analysis based on Nei’s genetic distance and a principal component analysis of isozyme frequencies did not detect a clear association between the geographic distribution of the landraces and their isozyme constitution. However, Nei’s genetic distance analysis clustered the bean landraces into two major groups which had a specific isozyme pattern, seed color, and seed size. The genetic analysis also detected a rare polymorphism for theMdh-2 locus, a null allele at theDiap-2 locus, and polymorphism for theAco-2 locus. The principal component analysis of isozyme frequencies showed that only 30% of the genotypes analyzed were similar to the Andean check and 5% of the samples were similar to Middle American check. This finding suggests a high frequency of hybridization between the Middle America and Andean gene pools in cultivated common bean from Chile.     

Genetic Relationship and Diversity of Four MangiferaSpecies Revealed through AFLP Analysis

We used AFLP analysis to explore the genetic relationship and diversity between and within 4 Mangifera species. We analyzed 35 accessions comprising 8 cultivars and 3 landraces of M. indica L., 11 landraces of M. odorata Griff., 7 landraces of M. foetida Lour., and 6 landraces of M. caesia Jack. Using 8 primer combinations produced a total of 518 bands, 499 (96.3%) of which were polymorphic among the 35 accessions. Clustering analysis showed that all 35 accessions were basically classified into 4 groups corresponding to the 4 Mangifera species. Our results indicate that the genetic relationship of these 4 Mangifera species based on AFLP analysis is in good agreement with their classification by classic methods. In addition, it was clearly revealed the genetic diversity between and within 4 Mangifera species. The findings obtained in this study are useful for the breeding in Mangifera species.     

Evaluation of Wild Lens Taxa for Agro-Morphological Traits, Fungal Diseases and Moisture Stress in North Western Indian Hills

Exploitation of wild gene pool for breeding is a common practice in an increasing number of cultivated plants. The cultivated lentil could not attain the substantial improvement in the yield potential due to loss of genes for higher productivity and lack of resistance against biotic and abiotic stresses. The absence of evaluation data of wild lentils for characters of economic importance, besides biotic and abiotic stresses, is one of the constraints in their use in lentil breeding programme. In the present study, 70 wild accessions from four wild Lens subsp./sp. (L. culinaris subsp. orientalis, L. odomensis, L. ervoides and L. nigricans) along with 3 checks (Precoz, PL-406 and PL-639) were evaluated for phenological and agro-morphological characters, for their reaction to three fungal diseases (wilt, powdery mildew and rust) and screened for tolerance to moisture stress. The wild accessions showed higher performance for branches/plant as compared to cultivated genotypes. Similarly, a few accessions of L. culinaris subsp. orientalis were earlier to flower and had higher seeds and seed yield/plant as compared to cultivated lentil. However, some were comparable with cultivated genotypes for flowers/peduncle, peduncle length and plant height. The mean performance for flowers per peduncle, leaflets per leaf, plant height, seeds and seed yield per plant increased, while decreased for days to flowering and maturity, and branches per plant during the evolution of cultivated lentil from the wild Lens taxa. Of Lens taxa, L. nigricans had the maximum resistant accessions for biotic and tolerance to abiotic stresses. The valuable variation existing among wild accessions can be exploited following introgression with cultivated lentils. It will help in the flow of useful genes from wild to cultivated lentil for generating wide spectrum of variability and its subsequent use in genetic restructuring of lentil.     

Genetic diversity among <Emphasis Type="Italic">Jatropha</Emphasis> species as revealed by RAPD markers

The genus Jatropha is native of tropical America with more than 200 species that are widely distributed in tropics with a promise for use as an oil crop for biodiesel. This investigation was carried out to assess the genetic diversity of 12 Jatropha species based on random amplified polymorphic DNA markers. From 26 random primers used, 18 primers gave reproducible amplification banding patterns of 112 polymorphic bands out of 134 bands scored accounting for 80.2% polymorphism across the genotypes. Three primers viz., OPA 4, OPF 11, and OPD 14 generated 100% polymorphic patterns. The polymorphic information content was highest for the primer OPD 14 (0.50) followed by the primers OPF 11 and OPAD 11 (0.48). Jaccard’s coefficient of similarity varied from 0.00 to 0.85, indicative of high level of genetic variation among the genotypes studied. UPGMA cluster analysis indicated three distinct clusters, one comprising all accessions of J. curcas L., while second included six species viz., J. ramanadensis Ramam., J. gossypiifolia L., J. podagrica Hook., J. tanjorensis J. L. Ellis et Saroja J. villosa Wight and J. integerrima Jacq. J. glandulifera Roxb. remained distinct and formed third cluster indicating its higher genetic distinctness from other species. The overall grouping pattern of clustering corresponds well with principal component analysis confirming patterns of genetic diversity observed among the species. The result provides valid guidelines for collection, conservation and characterization of Jatropha genetic resources.     

Biodiversity and ecogeography of wild Lactuca spp. in some European countries

Staff members of the Department of Botany of Palacký University in Olomouc and Gene Bank Department – Workplace Olomouc, Research Institute of Crop Production in Prague, Czech Republic, conducted an expedition in seven European countries (Austria, Czech Republic, France, Germany, Italy, the Netherlands, Switzerland) in August/September 1999 to collect wild Lactuca spp. germplasm and study its geographic distribution, ecology and biodiversity. During the mission, more than 600 locations were visited resulting in the collection of 602 seed samples (accessions) of wild Lactuca species and 13 seed samples of related genera (Chondrilla and Mycelis). Lactuca serriola f. serriola, L. serriola f. integrifolia, L. saligna and L. viminea subsp. chondrilliflora were prevalent in southern Europe (Italy, France), however, only L. serriola was common in central and western Europe (Austria, Czech Republic, Germany, Netherlands, Switzerland). The greatest diversity of Lactuca species was found in France, where also the most seed samples (165) were collected. The most characteristic habitats with a high density of Lactuca spp. populations were observed along roads and highways, grassy ditches, ruderal communities, and dust-heaps. Natural infections by powdery mildew (Erysiphe cichoracearum) and downy mildew (Bremia lactucae) on some wild Lactuca spp. were observed. Recent observations concerning the geographic distribution, population structure, habitats, and natural occurrence of diseases of Lactuca spp. are discussed. This assemblage of genetic resources of Lactuca spp. can serve as the basis of future studies of species diversification, spatial population structure, plant microevolution, domestication processes, and genetic variability of host-parasite interactions.     

Agricultural potential of Mediterranean grain and forage legumes: Key differences between and within Vicia species in terms of phenology, yield, and agronomy give insight into plant adaptation to semi-arid environments

In order to identify key adaptive traits which affect productivity in Mediterranean grain and forage legumes and simultaneously determine the agricultural potential of a wide range of Vicia species, germplasm collected from the wild throughout the eastern Mediterranean was grown under semi-arid conditions in Tel Hadya, Syria (313 mm growing season rainfall). These included species currently in use in Mediterranean agriculture, such as V. sativa L., as well as those more widely used in the past-such as V. ervilia L., but also a broad selection from Section Narbonensis (B. Fedtsch. ex Radzhi) Maxted, including V. narbonensis L, V. johannis Tamamsch., V. hyaeniscyamus Mout., V. serratifolia Jacq., and V. kalakhensis Khattab et al. V. faba, a near relative of the taxa in Section Narbonensis, was included as a domesticated control. Where applicable, a representative range of subspecies was used. Accessions were chosen from a wide range of habitats in terms of latitude (31.02–40.72 decimal^o), longitude (27.1–43.17 decimal^o), altitude (20–1510 m), rainfall (180–1700 mm/yr) and soil depth (5–50 cm) in order to maximise diversity within species. Agricultural potential was determined by measuring seed, hay and biological yield, as well as agronomic traits such as harvest index, standing crop height, and seed size. The comparative influence of phenology and key agronomic traits such as plant habit and seed size on productivity varied tremendously between species, depending on their reproductive strategies. In V. sativa and V. ervilia, the smaller seed species which rely on long vegetative phases and growing seasons to accumulate sufficient biomass to set seed, and in which there was comparatively little agronomic variation, phenology had a large impact on yield. In early emerging taxa such as V. ervilia and V. s. subsp. sativa, with built-in long vegetative phases and growing seasons, seed yield was negatively correlated with flower ing (r = –0.86 to –0.88), whereas the opposite was the case for later emerging taxa such as V. s. subsp. nigra (L.) Ehrh. (r = 0.95). Within V. narbonensis and relatives, the larger seeded Vicia species which rely on more conservative reproductive strategies where high seedling vigour associated with large seeds enables the species to enter reproductive phases relatively early, phenology had a much smaller impact on yield than did variation of key traits such as seed weight, plant habit and pod shattering. Among the undomesticated germplasm harvest indices ranged from 0.09–0.31, hay yields from 0.1–3.4 t/ha, seed yield from 0–2.0 t/ha, and dry matter at maturity from 1.6–6.5 t/ha. Sub-specific taxonomy was crucial in determining agronomic potential. V. narbonensis var. aegyptiaca Kornhuber ex Asch. et Schweinf. showed the most potential, combining an upright habit, large seeds (212 mg) and tendency to retain intact pods after maturity, with the highest yield, harvest index and crop height of all the wild Vicia species. V. sativa subsp. sativa, V. ervilia and V. narbonensis var. narbonensis were less productive, but still showed agricultural potential. The smaller seeded V. narbonensis, var. affinis, var. jordanica H. Schäf. and var. salmonea (Mout.) H. Schäf., and their close relatives V. johannis, V. hyaeniscyamus, V. serratifolia and V. kalakhensis have little to offer Mediterranean agriculture on the basis of poor agronomy.     

Use of GIS for Optimizing a Collecting Mission for a Rare Wild Pepper (<Emphasis Type="Italic">Capsicum flexuosum</Emphasis> Sendtn.) in Paraguay

This paper presents an effective method for prioritizing areas within a country for acquisition of germplasm of a crop gene pool for ex situ conservation. The method was applied to the rare wild pepper species, Capsicum flexuosum Sendtn., in south-east Paraguay. A model to prioritize areas for collecting germplasm was constructed by combining (1) a prediction of the species' geographic distribution based on the climate at previous collection points, (2) the distribution of forest margins (the species' natural habitat) and (3) areas accessible by road. The model was then tested in the field by visiting 20 sites having both high and low predicted probability of occurrence of C. flexuosum. Six new populations were found, representing a significant improvement over two previous collecting missions for the species in the same region, undertaken without the use of GIS targeting. Using the most optimistic analysis of model performance, C. flexuosum was found at five out of seven points predicted to harbour the species and not found at four of five points predicted not to harbour the species. The model was then improved by the use of higher resolution climate surfaces. It is recommended that future explorers use more recent and higher resolution satellite images to locate suitable habitats. The method is replicable for different species in different geographic regions and is offered as a means of optimizing efficiency in financially constrained, national plant genetic resources programs.     

Genetic diversity and conservation of two endangered eggplant relatives (<Emphasis Type="Italic">Solanum vespertilio</Emphasis> Aiton and <Emphasis Type="Italic">Solanum lidii</Emphasis> Sunding) endemic to the Canary Islands

Solanum vespertilio Aiton and Solanum lidii Sunding are endemic, endangered wild species from the Canary Islands. These species are of potential value for eggplant (S. melongena) breeding, given that they are part of the secondary genepool of this crop. We study genetic diversity with amplified fragment length polymorphisms (AFLPs) markers from 5 populations of S. vespertilio (47 samples) and 3 of S. lidii (26 samples). Five related African species (S. dasyphyllum Schumach. et Thonn., S. delagoense Dunal, S. campylacanthum Hochst., S. panduriforme E. Mey, S. aff. violaceum Ortega) were also included in the analysis. A total of 235 AFLP markers included 178 and 156 that were polymorphic in S. vespertilio and S. lidii, respectively. Analysis of genetic distance, phenograms, and principal component plots showed that these rare Canarian species are differentiated (G _ST = 0.412) from the continental materials and that Solanum vespertilio is more distinct to its African congeners than is S. lidii. There is a relatively high level of differentiation between the two species (G _ST = 0.373), that presumably reflects geographic restrictions (S. lidii to Gran Canaria; S. vespertilio essentially to Tenerife). However, both species have similar levels of total diversity. We speculate that the combination of the many unusual reproductive features (andromonoecy, zygomorphy, heteranthery and weak enantiostyly in S. vespertilio) help explain genetic diversity that is high for self compatible species. The high genetic diversity may also indicate populations were larger in the past. A decrease in population size could contribute to the relatively low genetic differentiation among the populations. The data presented herein provide the foundation for initiation of ex situ and in situ conservation programs for these wild relatives of eggplant. This paper is dedicated to the memory of Dr. Richard N. Lester, who made significant contributions to the taxonomy, biosystematics and conservation of genetic resources of African species of Solanum.     

Analysis of genetic diversity in Turkish sesame (Sesamum indicum L.) populations using RAPD markers⋆

The genetic diversity of 38 cultivated populations of Sesamum indicum L. from four different regions of Turkey was estimated at the DNA level with the random amplified polymorphic DNA (RAPD) technique. Sixty-one bands were obtained for all populations 78% of which were polymorphic. Analysis of molecular variance (AMOVA) was used to investigate the genetic diversity of the populations which yielded highly significant differences among populations within regions (91.9% of the total genetic diversity). According to AMOVA and Shannon's index that were performed separately for each region, the highest value of genetic variation was observed among Northwest region populations (CV = 7.7; H₀ = 0.304) and lowest in the Southeast regions' populations (CV = 2.6; H₀ = 0.068). Nei and Li's similarity index was calculated and phylogenetic tree was established using the neighbor-joining algorithm. This phenetic analysis grouped 35 of 38 accessions in six groups leaving three highly diverse accessions outside. Wagner phylogenetic method was used to assess the phylogenetic relationships among the populations. In the majority-rule consensus tree, only 7 of the 32 forks showed above 60% occurrence. Using Principal Coordinate Analysis (PCO) of the RAPD data set, the groups were clearly separated along the first three axis. These results indicate that RAPD technique is useful for sesame systematics, and should be valuable for the maintenance of germplasm banks and the efficient choice of parents in breeding programs.     

Genetic structure among earthworms (Lumbricus terrestris L.) from different sampling sites in western Germany based on random amplified polymorphic DNA

Earthworms are being used as bio-indicators to assess terrestrial pollution. However, it is often not known whether their populations possess a uniform genetic structure, which would allow comparison of residues or biological properties of earthworms from different sampling locations. In order to investigate this point, random amplified polymorphic DNA (RAPD) variation was surveyed in earthworms (Lumbricus terrestris) from five different sampling sites in Germany. Forty oligonucleotide RAPD primers (10 base pairs in length) were screened, three of which produced high polymorphic band patterns. A total of 61 DNA fragments were detected in 90 individuals of L. terrestris from five sampling sites with 49 (80.3%) RAPD markers being polymorphic. The genetic similarities within (band sharing rates between 0.756 and 0.795) and among the L. terrestris populations (0.635) were similar even at widely separate locations. Inter-population variation in the RAPD pattern for all five earthworm populations accounted for 37.9% of the total variation, while intra-population variation for three adjacent Saarland populations accounted for only 18.0% of the total variation. Principal component analysis (PCA) and the genetic distances of the populations confirm these results. Twenty-four percent of the genetic distance is caused by geographical isolation as shown by a test for isolation by distance. These results show that L. terrestris fulfils the genetic qualifications for a bio-indicator particularly at closely located sampling sites. However, the results also suggest that earthworm studies of widely separated locations should include genetic characterisation of the earthworm samples.