The abundance and efficacy of Rhizobium leguminosarum bv. viciae in cultivated soils of the eastern Canadian prairie

For optimum production, the use of commercial rhizobial inoculant on pea (Pisum sativum L.) at seeding is necessary in the absence of compatible rhizobial strains or when rhizobial soil populations are low or symbiotically ineffective. Multiple site experiments were conducted to characterize the abundance and effectiveness of resident populations of Rhizobium leguminosarum bv. viciae (Rlv) in eastern Canadian prairie soils. A survey of 20 sites across a broad geographical range of southern Manitoba was carried out in 1998 and was followed by more intensive study of five of the sites in 1999 and 2000. Appreciable nodulation of uninoculated pea was observed at all sites which had previously grown inoculated pea. However, uninoculated pea grown at two sites, which had not previously grown pea, had negligible nodulation. Likewise, wild Lathyrus sp. and Vicia sp. plants collected from uncultivated areas adjacent to agricultural sites were poorly nodulated. In the more intensively studied sites, there was a tendency towards higher nodulation in pea plants receiving commercial inoculant containing Rlv strain PBC108 across all site-years (e.g., 4.7% in nodulation and 22% in nodule mass), but the effect was significant at only 2 of 10 site-years. Despite a relatively high range of soil pH (6-8), regression analysis indicated that decreasing soil pH resulted in lower nodulation rates. Likewise, electrical conductivity (EC) was correlated to nodulation levels, however the effect of EC was likely more indicative of the influence of soil texture and organic matter than salinity. As with nodulation, commercial inoculation tended to increase above-ground dry matter (DM) and fixed-N (estimated by the difference method) at the early pod-filling stage, but again the effects were significant at only 2 of 10 site-years. Specifically, above-ground DM and fixed-N levels were up to 29 and 51% greater, respectively, in inoculated compared to non-inoculated treatments at these sites. Addition of N-fertilizer at a rate of 100 kg N ha⁻¹ decreased nodulation at almost all site-years (by as much as 70% at one site), but rarely resulted in increases in above-ground DM compared to inoculated plots. The study indicates for the first time that populations of infective, and generally effective strains of Rlv occur broadly in agricultural soils across the eastern Canadian prairie, but that there is a tendency for increased symbiotic efficiency with the use of commercial inoculant.     

Size, symbiotic effectiveness and genetic diversity of field pea rhizobia (Rhizobium leguminosarum bv. viciae) populations in South Australian soils

Field pea (Pisum sativum L.) is widely grown in South Australia (SA), often without inoculation with commercial rhizobia. To establish if symbiotic factors are limiting the growth of field pea we examined the size, symbiotic effectiveness and diversity of populations of field pea rhizobia (Rhizobium leguminosarum bv. viciae) that have become naturalised in South Australian soils and nodulate many pea crops. Most probable number plant infection tests on 33 soils showed that R. l. bv. viciae populations ranged from undetectable (six soils) to 32×10³ rhizobia g⁻¹ of dry soil. Twenty-four of the 33 soils contained more than 100 rhizobia g⁻¹ soil. Three of the six soils in which no R. l. bv. viciae were detected had not grown a host legume (field pea, faba bean, vetch or lentil). For soils that had grown a host legume, there was no correlation between the size of R. l. bv. viciae populations and either the time since a host legume had been grown or any measured soil factor (pH, inorganic N and organic C). In glasshouse experiments, inoculation of the field pea cultivar Parafield with the commercial Rhizobium strain SU303 resulted in a highly effective symbiosis. The SU303 treatment produced as much shoot dry weight as the mineral N treatment and more than 2.9 times the shoot dry weight of the uninoculated treatment. Twenty-two of the 33 naturalised populations of rhizobia (applied to pea plants as soil suspensions) produced prompt and abundant nodulation. These symbioses were generally effective at N₂ fixation, with shoot dry weight ranging from 98% (soil 21) down to 61% (soil 30) of the SU303 treatment, the least effective population of rhizobia still producing nearly double the growth of the uninoculated treatment. Low shoot dry weights resulting from most of the remaining soil treatments were associated with delayed or erratic nodulation caused by low numbers of rhizobia. Random amplified polymorphic DNA (RAPD) polymerase chain reaction (PCR) fingerprinting of 70 rhizobial isolates recovered from five of the 33 soils (14 isolates from each soil) showed that naturalised populations were composed of multiple (5-9) strain types. There was little evidence of strain dominance, with a single strain type occupying more than 30% of trap host nodules in only two of the five populations. Cluster analysis of RAPD PCR banding patterns showed that strain types in naturalised populations were not closely related to the current commercial inoculant strain for field pea (SU303, ≥75% dissimilarity), six previous field pea inoculant strains (≥55% dissimilarity) or a former commercial inoculant strain for faba bean (WSM1274, ≥66% dissimilarity). Two of the most closely related strain types (≤15% dissimilarity) were found at widely separate locations in SA and may have potential as commercial inoculant strains. Given the size and diversity of the naturalised pea rhizobia populations in SA soils and their relative effectiveness, it is unlikely that inoculation with a commercial strain of rhizobia will improve N₂ fixation in field pea crops, unless the number of rhizobia in the soil is very low or absent (e.g. where a legume host has not been previously grown and for three soils from western Eyre Peninsula). The general effectiveness of the pea rhizobia populations also indicates that reduced N₂ fixation is unlikely to be the major cause of the declining field pea yields observed in recent times.     

Transfer and loss of naturally-occurring plasmids among isolates of Rhizobium leguminosarum bv. viciae in heavy metal contaminated soils

Plasmid transfer among isolates of Rhizobium leguminosarum bv. viciae in heavy metal contaminated soils from a long-term experiment in Braunschweig, Germany, was investigated under laboratory conditions. Three replicate samples each of four sterilized soils with total Zn contents of 54, 104, 208 and 340 mg kg⁻¹ were inoculated with an equal number (1×10⁵ cells g⁻¹ soil) of seven different, well-characterized isolates of R. leguminosarum bv. viciae. Four of the isolates were from an uncontaminated control plot (total Zn 54 mg kg⁻¹) and three were from a metal-contaminated plot (total Zn 340 mg kg⁻¹).After 1 year the population size was between 10⁶ and 10⁷ g⁻¹ soil, and remained at this level in all but the most contaminated soil. In the soil from the most contaminated plot no initial increase in rhizobial numbers was seen, and the population declined after 1 year to <30 cells g⁻¹ soil after 4 years. One isolate originally from uncontaminated soil that had five large plasmids (no. 2-8-27) was the most abundant type re-isolated from all of the soils. Isolates originally from the metal-contaminated soils were only recovered in the most contaminated soil. After 1 year, four isolates with plasmid profiles distinct from those inoculated into the soils were recovered. One isolate in the control soil appeared to have lost a plasmid. Three isolates from heavy metal contaminated soils (one isolate from the soil with total Zn 208 mg kg⁻¹ and two isolates from the soil with total Zn 340 mg kg⁻¹) had all acquired one plasmid. Plasmid transfer was confirmed using the distinct ITS-RFLP types of the isolates and DNA hybridization using probes specific to the transferred plasmid. The transconjugant of 2-8-27 which had gained a plasmid was found in one replicate after 2 years of the most contaminated soil but comprised more than 50% of the isolates. A similar type appeared in a separate replicate of the most contaminated soil after 3 years and persisted in both of these soils until the final sampling after 4 years. After 2 years isolates were recovered from four of the soil replicates with the chromosomal type of 2-8-27 which appeared to have lost one plasmid, but these were not recovered subsequently.Isolate 2-8-27 was among the isolates most sensitive to Zn in laboratory assays, whereas isolate 7-13-1 showed greater zinc tolerance. Acquisition of the plasmid conferred enhanced Zn tolerance to the recipients, but transconjugant isolates were not as metal tolerant as 7-13-1, the putative donor. Laboratory matings between 2-8-27 and 7-13-1 in the presence of Zn resulted in the conjugal transfer of the same small plasmid from 7-13-1 to isolate 2-8-27 and the transconjugant had enhanced metal tolerance. Our results show that transfer of naturally-occurring plasmids among rhizobial strains is stimulated by increased metal concentrations in soil. We further demonstrate that the transfer of naturally-occurring plasmids is important in conferring enhanced tolerance to elevated zinc concentrations in rhizobia.     

Distribution and efficiency of Rhizobium leguminosarum strains nodulating Phaseolus vulgaris in Northern Spanish soils: Selection of native strains that replace conventional N fertilization

Phaseolus vulgaris is a legume extensively cultivated in Spain, León province being the most important producer. This province produces selected varieties of common bean highly appreciated by their quality that warrants a Protected Geographic Indication (PGI). In this work we analysed the rhizobia present in nodules of the variety “Riñón” in several soils from León province in order to select native rhizobial strains to be used as biofertilizers. The analysis of rrs and housekeeping genes of these strains showed that they belong to two phylogenetic groups within Rhizobium leguminosarum (I and II). Although the group II strains were most abundant in nodules, very effective strains were also found in group I. Strains LCS0306 from group I and LBM1123 from group II were the best nitrogen fixers among all strains isolated and were selected for field experiments. The field research showed that the biofertilization of common bean with native and selected rhizobial strains can completely replace the fertilization with chemical N fertilizers. The biofertiliser designed in such way, was valid for the whole agroecological area, regardless the specific properties of each soil and microclimatic conditions. This conclusion can be generalised as a strategy for the development of biofertilisers in different agroecological conditions worldwide.     

Physiological responses to acid stress of an acid-soil tolerant and an acid-soil sensitive strain of Rhizobium leguminosarum biovar trifolii

Physiological responses to acid stress in two strains of Rhizobium leguminosarum bv trifolii of differing acid-soil tolerance were compared. Acidity affected the size and morphology of the acid-tolerant strain, WSM409, but not of the acid-sensitive strain, TA1. Acid grown cells of WSM409 and TA1 had less cell-associated Ca and Mg and more P than cells grown at pH 7.0. Potassium content was lower in acid grown cells; WSM409 was less affected by pH than that in TA1. WSM409 was more tolerant of pH shock at pH 3.5 when grown at pH 4.8 than when grown at pH 7.0. TA1 was more sensitive to pH shock when grown at pH 4.8 than when grown at pH 7.0. WSM409 shows a characteristic adaptive acid tolerance response, whereas TA1 shows an acid sensitive response.     

Rapid identification and discrimination among Egyptian genotypes of Rhizobium leguminosarum bv. viciae and Sinorhizobium meliloti nodulating faba bean (Vicia faba L.) by analysis of nodC, ARDRA, and rDNA sequence analysis

Twenty-eight Rhizobium strains were isolated from the root nodules of faba bean (Vicia faba L.) collected from 11 governorates in Egypt. A majority of these strains (57%) were identified as Rhizobium leguminosarum bv. viciae (Rlv) based on analysis of a nodC gene fragment amplified using specific primers for these faba bean symbionts. The strains were characterized using a polyphasic approach, including nodulation pattern, tolerance to environmental stresses, and genetic diversity based on amplified ribosomal DNA-restriction analysis (ARDRA) of both 16S and 23S rDNA. Analysis of tolerance to environmental stresses revealed that some of these strains can survive in the presence of 1% NaCl and a majority of them survived well at 37 °C. ARDRA indicated that the strains could be divided into six 16S rDNA genotypes and five 23S rDNA genotypes. Sequence analysis of 16S rDNA indicated that 57% were Rlv, two strains were Rhizobium etli, one strain was taxonomically related to Rhizobium rubi, and a group of strains were most closely related to Sinorhizobium meliloti. Results of these studies indicate that genetically diverse rhizobial strains are capable of forming N₂-fixing symbiotic associations with faba bean and PCR done using nodC primers allows for the rapid identification of V. faba symbionts.     

Quantitative PCR assays to enumerate Rhizobium leguminosarum strains in soil also target non viable cells and overestimate those detected by the plant infection method

The plant infection method is commonly used to estimate the Most Probable Number (MPN) of soil rhizobia. Here, a qPCR method was set-up and validated with newly developed ANU (strain specific) and RHIZ (more general) primers to quantify the specific Rhizobium leguminosarum bv. trifolii ANU843 strain or general R. leguminosarum strains. Detection limits of qPCR protocols in soil were 1.2 × 10⁴ (ANU) and 4.2 × 10³ (RHIZ) cells per g soil. The qPCR assay appears robust and accurate in freshly inoculated soils but overestimated MPN for indigenous soil rhizobia. An incubation experiment showed that qPCR detected added DNA or non viable cells in soils up to 5 months after addition and incubation at 20 °C in moist conditions.     

Competitive abilities of common field isolates and a commercial strain of Rhizobium leguminosarum bv. trifolii for clover nodule occupancy

We previously reported that commercial Rhizobium leguminosarum bv. trifolii inoculants failed to outcompete naturalized strains for nodule occupation of clover sown into an alkaline soil [Aust. J. Agric. Res. 53 (2002) 1019]. Two field isolates that dominated nodule occupancy at the field site were labeled with a PnifH-gusA marker. Marked strains were chosen on the basis that they were equally competitive and fixed similar amounts of nitrogen in comparison to their parental strain. The minitransposon insertions were cloned and sequence analysis revealed that neither lesion disrupted the integrity of any known gene. The marked strains were then used to follow nodule occupancy of Trifolium alexandrinum in competition against the commercial inoculant TA1 under a range of experimental conditions. In co-inoculation experiments in sand-vermiculite, TA1 outcompeted each marked field isolate for nodule occupancy. However, using TA1-inoculated seed sown into alkaline soil containing a marked field strain, it was demonstrated that by increasing the cell number of marked rhizobia in the soil and reducing the cell number of the commercial inoculant, the proportion of nodules occupied by TA1 was reduced. These studies indicate that the ability of the field isolates to dominate nodule occupancy in the alkaline field soils was most likely caused by poor commercial inoculant survival providing the advantage for naturalized soil rhizobia to initiate nodulation.     

Distribution and genetic diversity of rhizobia nodulating natural populations of Medicago truncatula in tunisian soils

A collection of 299 isolates of rhizobia nodulating Medicago truncatula was isolated from 10 Tunisian soils and was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR/RFLP) of 16S rRNA gene. Results showed that 227 and 72 isolates were assigned, respectively, to Sinorhizobium meliloti and Sinorhizobium medicae. In 9 out of 10 soils S. meliloti was detected, whereas S. medicae was recovered from only 5 out of 10 soils. The cross-nodulation of three populations of M. truncatula grown on Bulla Regia soil, which contained naturally the two Sinorhizobium species, showed that M. truncatula population collected from Amra site was selective to S. meliloti at least in soil conditions. Forty-eight isolates of each Sinorhizobium species trapped by M. truncatula populations collected from Bulla Regia, Soliman and Rhayet sites on Bulla Regia soil were characterized by repetitive extragenic palindromic-PCR (REP-PCR) and showed a clear distinction between the two Sinorhizobium species and a higher diversity for S. meliloti.     

Polyphasic characterization of Brazilian Rhizobium tropici strains effective in fixing N2 with common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris) is native to the Americas, and Rhizobium etli is the dominant microsymbiont in both the Mesoamerican and the Andean centers of genetic diversification. Wild common beans are not found in Brazil, although the legume has been cropped in the country throughout time and all but one of the rhizobial species that nodulate it (Rhizobium gallicum) have been broadly detected in Brazilian soils. However, the majority of the effective rhizobial strains isolated so far from field-grown plants belong to R. tropici. This study describes the analysis of symbiotic and non-symbiotic genes of 15 effective R. tropici strains, isolated from four geographically distant regions in Brazil. With RFLP-PCR of the 16S and 23S rRNA genes and sequence analysis of 16S rRNA, two clusters were observed, one related to R. tropici type A and another to type B strains. Diversity in ribosomal genes was high, indicating that type A strains might represent a new species. High intraspecies diversity was also observed in the rep-PCR analysis with BOX, ERIC and REP primers. However, in the RFLP-PCR analysis of nifH and nodC genes, all R. tropici showed unique combinations of profiles, which might reflect an evolutionary strategy to maximize N₂ fixation.     

Genetic diversity of indigenous common bean (Phaseolus vulgaris) rhizobia in two Brazilian ecosystems

Although rhizobia for common bean (Phaseolus vulgaris L.) are established in most Brazilian soils, understanding of their genetic diversity is very poor. This study characterized bean strains from two contrasting ecosystems in Brazil, the Northeast Region, with a semi-arid climate and neutral soils and the South Region, with a humid subtropical climate and acid soils. Seedlings of the cultivars Negro Argel and Aporé were used to trap 243 rhizobial isolates from 12 out of 14 sites. An analysis of ERIC-PCR products revealed enormous variability, with 81% of the isolates representing unique strains considering a level of 70% of similarity. In general, there was no effect of either the bean cultivar, or the ecosystem on rhizobial diversity. One-hundred and one strains showing genetic relatedness (ERIC-PCR) less than 70% were further analyzed using restriction fragment length polymorphism (RFLP) of the 16 S rDNA cleaved with five restriction enzymes. Twenty-five different profile combinations were obtained. Rhizobium etli was the predominant species, with 73 strains showing similar RFLP profiles, while 12 other strains differed only by the profile with one restriction enzyme. Fifty strains were submitted to sequencing of a 16 S rDNA fragment, and 34 clustered with R. etli, including strains with RFLP-PCR profiles similar to those species or differing by one restriction enzyme. However, other strains differing by one or two enzymes were genetically distant from R. etli and two strains with identical profiles showed higher similarity to Sinorhizobium fredii. Other strains showed higher similarity of bases with R. tropici, R. leguminosarum and Mesorhizobium plurifarium, but some strains were quite dissimilar and may represent new species. Great variability was also verified among the sequenced strains in relation to the ability to grow in YMA at 40 °C, in LB, to synthesize melanin in vitro, as well as in symbiotic performance, including differences in relation to the described species, e.g. many R. etli strains were able to grow in LB and in YMA at 40 °C, and not all R. tropici were able to nodulate Leucaena.     

Effect of selective logging on the genetic diversity of Scaphium macropodum

We examined the effect of selective logging on the genetic diversity of Scaphium macropodum using RAPD markers via two approaches: (1) to investigate the immediate effect by studying a same population before and after logging, and (2) to determine the long term effect by comparing two regenerated stands with an adjacent unlogged stand, assuming that they were genetically identical before logging. Results showed no negative immediate impact for the first approach, probably due to the high abundance and heterogeneity of S. macropodum in the compartment investigated. However, for the latter approach, substantial genetic erosion (i.e. 31.5% reduction for Shannon diversity, H) was detected in one of the regenerated stands corresponding to its extremely low tree density for S. macropodum. This implies the possible occurrence of genetic drift and increased inbreeding due to population decline as a result of logging. However, the observed genetic differences among the three sub-populations having prevailed before logging cannot be totally discounted in the second approach. This study also demonstrates the use of tree density as a good surrogate measure of genetic diversity. The present harvesting system in Malaysia based on a general cutting limit need to be refined; the basis for determining cutting limit in a forest management unit should consider abundance of commercial species.     

Molecular markers and a sequence deletion in intron 2 of the putative partial homologue of LEAFY reveal geographical structure to genetic diversity in the acutely threatened legume genus Clianthus

Clianthus is an acutely threatened, bird-pollinated genus endemic to New Zealand, represented in the wild by only one population of C. puniceus and 11 populations of C. maximus, each with very few individuals (typically <10 per population). A limited number of named Clianthus cultivars of indeterminate origin are commonly grown as ornamentals. Genomic DNA from individual Clianthus plants was extracted for genetic diversity analysis using a range of molecular markers, including amplified fragment length polymorphism (AFLP). Data were analysed by the unweighted pair-group method with arithmetic averaging (UPGMA), the generation of Neighbor-Joining trees, and analyses of molecular variance (AMOVA). Genetic distance between wild populations of C. maximus was highly correlated with geographical distance between populations. Sequencing of intron 2 of a putative partial homologue of the floral meristem identity gene LEAFY (CmLFY) revealed a 7 bp deletion that was exhibited homozygously in the more northern populations of C. maximus, and in all individuals tested from the sole population of C. puniceus. This deletion was not exhibited in more southern populations of C. maximus. Further, one geographically intermediate population contained some plants that were heterozygous for the deletion. Parallel analyses of cultivated Clianthus genotypes, more than half of which were also homozygous for the 7 bp deletion, showed that these were not representative of the broad, but threatened, diversity remaining in the wild. It is argued that wild populations of C. maximus are unlikely to have arisen from the escape of plants from cultivation. Conservation effort should focus on the protection and study of the extant plants in these wild populations, rather than on the introduction of disturbance regimes to uncover potential seed banks.     

Genetic diversity within Aspergillus flavus strains isolated from peanut-cropped soils in Argentina

The genetic diversity of Aspergillus flavus populations isolated from the peanut-cropped soils in the peanut-growing region at Cordoba Province was evaluated by analysis of vegetative compatibility group (VCG). VCG_s were determined through complementation assays between nitrate-nonutilizing (NNO) mutants. Fifty-six VCG_s were identified from 100 isolates. Twenty-five VCG_s contained two or more isolates and 31 VCG_s contained only a single isolate. In general, there were significant differences among VCG_s in aflatoxin and CPA production. One VCG group included a single atoxigenic strain since it was neither aflatoxin nor cyclopiazonic acid producer. This isolate could be useful as a biological control agent, since it was unable to form a stable heterokaryon in the complementation test with the other isolates. Seven A. flavus isolated from soil were atypical because they simultaneously produced aflatoxins B, G and CPA.     

High genetic diversity and heterogeneous parasite load in the earthworm Lumbricus terrestris on a German meadow

In parasite-host dynamics, parasites exert frequency-dependent selection on their hosts by favouring rare alleles that may confer resistance against infection. Therefore host populations that suffer strong parasite stress should maintain higher levels of genetic variability. We studied the Lumbricus terrestris-Monocystis sp. host-parasite system at a microgeographical scale. Using three polymorphic microsatellite loci on one large earthworm population sampled at 26 different sites (281 genotypes), we tested the relationship between parasite load and genetic variation in natural samples of the common earthworm L. terrestris. Our analysis yielded the following: (1) parasite load varied significantly across sites in this population; (2) there was no consistent evidence for heterozygote deficiency (observed heterozygosities ranged between 0.74 and 0.87), indicating a low level of inbreeding; (3) there was no significant genetic structuring among sample sites; (4) we could not identify a significant association between parasite load and population genetic diversity; (5) there was considerable population differentiation (15.17%) between our German samples and a Canadian L. terrestris reference population. Our study provides insight into the population genetics of one of the most economically important soil organisms on a microgeographic scale.     

Competitiveness of Rhizobium leguminosarum bv. phaseoli strains in relation to environmental stress and plant defense mechanisms

Summary Six Rhizobium leguminosarum bv. phaseoli strains (Ciat 151, Ciat 895, Ciat 899, CE3, H2C, Kim5s) were tested for nodule occupancy in different bean cultivars at two field sites (one fertile, one acid tropical soil) and in the greenhouse. The effects of several environmental factors such as low pH, high temperature, Al and Mn toxicity, iron deficiency, bean tannins, and bean phytoalexins were tested in vitro. Strain Kim5s was competitive under all tested conditions while strains CE3 and H2C had consistently low nodule occupancy levels. Strain Ciat 151 was superior to the other inoculant strains in the acid soil but competed poorly in the fertile soil. Strain Ciat 895 was more competitive in the fertile soil. There was a decline in nodule occupancy for all strains tested from the first trifoliate leaf stage to the pod-filling stage. No plant genotype effect on nodule occupancy was observed. There were significant (P<0.05) plant genotype and location effects, but no significant strain effect on acetylene reduction activity, plant dry weight, and nodule number. The greenhouse experiments confirmed, at least partially, the results from the field trials. In Leonard jars with an acid soil, strains Ciat 151 and Kim5s were amongst the most competitive strains. In coinoculation experiments, Kim5s was the most competitive strain, followed by Ciat 899 and Ciat 895. The competitiveness of a given strain was affected by the coinoculant strain. Tolerance in vitro to low pH, high growth temperature, Al or Mn toxicity, or Fe limitation was not related to competitiveness of the inoculum strains. The sensitivity of the strains towards bean tannins or bean phytoalexins also was not correlated with their competitiveness.     

Relationship between population size and genetic diversity in endangered populations of the European bullhead (Cottus gobio): implications for conservation

This study investigated the relationship between the current size of endangered bullhead (Cottus gobio) populations and microsatellite genetic variability. Additionally, the microsatellite data were used to evaluate whether a genetic test for population bottlenecks was able to provide evidence of recent severe population declines. Finally, our results were used to develop conservation priorities and measures. Population size appears to be a crucial parameter in determining the amount of genetic diversity that can be preserved in bullheads, since a significant positive correlation was observed between both variables. Furthermore, in some populations we were able to detect genetic signatures of the documented decline in population size. We suggest that the most immediate goal for bullhead conservation should be to increase the size and the range of the populations, and in doing so minimise or even reverse further genetic erosion. Potential management actions like habitat quality improvement, reduction of river fragmentation and supplementation programmes (translocation, supportive breeding) are discussed.     

MtDNA genetic diversity and population history of a dwindling raptorial bird, the red kite (Milvus milvus)

The red kite (Milvus milvus) occurs in a relatively small area in the southwestern Palearctic region, with population strongholds in Central Europe. Following strong human persecutions at the beginning of the 20th century, populations have receded, particularly in peripheral areas and islands. In order to describe and compare levels of genetic diversity and phylogeographic patterns throughout its entire distribution in Europe, sequence variation of a 357 bps part of the mitochondrial DNA control region was assessed in eight populations and 105 individuals. Overall, results indicate that population declines have affected red kite mtDNA variation. We found low levels of genetic diversity (values of nucleotide diversity ranging from 0 in Majorca island to 0.0062 in Central Europe), with only 10 distinct haplotypes, separated by low levels of genetic divergence (mean sequence divergence = 0.75%). Highest haplotype and nucleotide diversities match with demographic expectations, and were found in Central European and Central Spanish samples, where present strongholds occur, and lowest values in the declining southern Spanish and insular samples. Φst estimates indicated moderate gene flow between populations. Phylogeographic patterns and mismatch distributions analyses suggest central European regions may have been colonized from southern glacial refugia (in the Italian or Iberian peninsulas). Interspecific phylogenetic comparisons and divergence date estimates indicated the genetic split between the red kite and its closely related species, the black kite (Milvus migrans), might be relatively recent. The low level of genetic variation found in the red kite mitochondrial control region, compared to the black kite, is likely the result of relatively recent divergence (associated with founder events), successive bottlenecks and small population sizes. As there are several ongoing projects aimed at reinforcing populations in countries such as the United Kingdom, Italy or Spain, our results may prove useful for the genetic management of the species.     

Plantations of Convallaria majalis L. as a threat to the natural stands of the species: Genetic variability of the cultivated plants and natural populations

The level of polymorphism, genetic variability and relatedness of Convallaria majalis-populations (species native in Poland, under partial legal protection) obtained from three Polish regions and from commercial producers (Polish and Dutch) were studied. In addition the differences between the cultivated plants and those occurring in natural stands were analyzed. Each region was represented by at least 20 populations among which half were collected in natural stands and half from cultivation sites (botanical gardens, private gardens and cemeteries), and compared with samples obtained directly from commercial producers. Seven primer pairs used for AFLP profiling amplified 466 scoreable DNA fragments that were used for multidimensional scaling and clustering. The above analyses make it possible to clearly distinguish among individuals and revealed groups of populations according to their geographic origin. Samples from populations collected in natural stands and cultivated in the same region did not differ from each other significantly.These results suggested that cultivated plants were probably obtained directly from the natural stand and the influence of plant cultures on natural populations was rather small.