Genetic variation in populations of the endangered fish Ladigesocypris ghigii and its implications for conservation

1. The genetic variation of the endangered freshwater fish Ladigesocypris ghigii, endemic to the island of Rhodes (Greece), was investigated for nine populations, originating from seven different stream systems and a reservoir, both at the mtDNA and nuclear level, in order to suggest conservation actions. 2. Both restriction fragment length polymorphism analysis of five segments of mitochondrial DNA (ND‐5/6, COI and 12S‐16S rRNA) amplified by polymerase chain reaction, and random amplified polymorphic DNA analysis, revealed extremely low levels of intra‐population polymorphism. It is highly likely that the low intra‐population variability is the result of successive bottleneck events evident in shrinkage and expansion of the populations year after year, which may have led to a complete loss of several genotypes and haplotypes, and an increased degree of inbreeding. 3. Inter‐population genetic structuring was high, with fixation of haplotypes within six of the nine populations and fixation of alleles within populations originating from different waterbodies. It is probable that all haplotypes and/or alleles found were initially represented in all populations. However, because of the long time of isolation coupled with successive bottleneck and subsequent genetic drift, common mtDNA haplotypes and alleles among the populations may have become rare or extinct through stochastic lineage loss. 4. Although nucleotide divergence among haplotypes was very shallow, half of the haplotypes recorded (three of six), resulted from nucleotide changes on the 12S–16S rRNA segments, which are the most conserved part of the mitochondrial genome. This fact may indicate that the observed genetic variation did not necessarily result only from the retention of ancestral polymorphism, but may have arisen through mutation and complete lineage sorting over a relatively small number of generations, once the populations had become isolated from one another. 5. Our data suggest that two of the L. ghigii populations may be on independent evolutionary trajectories. Considering that each population appears so far well adapted within each site, all populations should be managed and conserved separately.     

Inverse Correlation between ABA Content and Germinability throughout the Maturation and the In Vitro Culture of the Embryo of Phaseolus vulgaris

Prevost, I. and Le Page-Degivry, M. Th. 1985. Inverse correlationbetween ABA content and germinability throughout the maturationand the in vitro culture of the embryo of Phaseolus vulgaris.—J.exp. Bot. 36: 1457–1464. Changes in embryo abscisic acid (ABA) content during the maturationof the seed of Phaseolus vulgaris cv. Contender were followed,using a radio-immunoassay. The pattern of change is similarto that already described in several species: a rapid increase(from the 18th to 29th day after anthesis), was followed bya decrease, the ABA level being ten times lower on the 48thday than on the 29th. Embryos isolated from the 18th to the48th day after anthesis were able to ‘germinate’when cultivated on a mineral medium supplemented with sucroseand agar. The development pattern varied throughout the embryogenesisand could be correlated with the differentiation of the embryoat the time of isolation. Before germination could take place,we observed a lag phase, the duration of which could be correlatedwith embryo ABA content. As ABA content increased in the youngestembryos the duration of the lag phase increased. In the sameway, the number of days to germination was shown to diminishas ABA content decreased. Inverse correlation between ABA contentand germinability was thus demonstrated throughout the developmentof the embryo. During in vitro culture, free ABA content decreased in the embryoand reached low values a few days before germination occurred.So the beginning of root elongation in culture was again wellcorrelated with the disappearance of free endogenous ABA. Atransfer experiment inducing an earlier germination associatedwith a more limited development suggests that the lag phaseis associated with an active continuation of embryonic development Key words: Embryo maturation, abscisic acid, germinability     

The Import and Redistribution of Several Cations and Anions in Tomato Leaves

Wolterbeek, H. Th. and De Bruin, M. 1986. The import and redistributionof several cations and anions in tomato leaves.—J. exp.Bot. 37: 331–340. The upward movements in the xylem and redistribution from theleaf of Na⁺ , K⁺ , Rb⁺, Cs⁺ and four anions were examined insub-systems of tomato plants (Lycopersicon esculentum, Mill.cv. Tiny Tim). There was a delay with respect to the redistributionof newly imported elements from the source leaf of about 16–20h for all four alkali ions. This is considerably less than theapparent delay for the anions Sb(SO₄)^– WO₄^2– Mo₇O₂₄^6–and AsO₄^3– The prolonged delay for the anions is suggestedto be a consequence of metabolic transformation in the leaf.Reduction of the source-sink activity ratio did not decreasethe delay period from the source leaf, but apparently causedincreased Na⁺ transfer from the xylem. It is concluded thatthe application of a detailed mathematical descnption of upwardelement movement has considerable potential possibilities forunderstanding circulation of nutrients in the plant. Key words: Alkali ions, anions, xylem, phloem, redistribution, tomato     

Relationships between Adsorption, Chemical State and Fluxes of Cadmium Applied as Cd(NO3)2 in Isolated Xylem Cell Walls of Tomato

Wolterbeek, H. Th. 1987. Relationships between adsorption, chemicalstate and fluxes of cadmium applied as Cd(NO₃)₂ in isolatedxylem cell walls of tomato.—J. exp. Bot. 38: 419–432. Isolated xylem cell wall pieces were applied as membranes inion diffusion experiments. The cell walls were isolated fromtomato internodes (Lycopersicon esculentum Mill, cv. Tiny Tim)and sealed in a two-compartment diffusion system. In flux andadsorption calculations, the cell wall was regarded as a leakymembrane with parallel fluxes through Donnan Free Space (DFS)and Water Free Space (WFS). During the experiments absorptioninto and diffusion across the walls was determined of Cd^{2 +}, applied as ¹¹⁵Cd(NO₃)₂. Flux experiments with ⁸²Br^–indicated that excluded volume effects and path tortuosity resultedin apparent WFS diffusion coefficients in the walls which were0·012 times as high as in water. The free proton concentration in the DFS was shown to be relatedto a complex formation between fixed charges and Cd^{2 +}. Thecell wall permeability for Cd^{2 +} and NO₃^– varied withapplied and absorbed concentrations, and the Cd^{2 +} flux curveshowed an inflexion point coinciding with a buffered degreeof dissociation of fixed charges in the DFS. The necessary couplingof fluxes of opposite charges resulted in relatively high NO₃^–and small Cd^{2 +} permeability of the DFS for strongly dilutedsolutions (P = 10^–4 m s^–1 and 10^–11 m s^–1for NO₃^– and Cd^{2 +} respectively). The results demonstratethe possible regulatory effects of the cell wall in processesof ion transfer from xylem vessels, or ion uptake in plant tissues. Key words: Cadmium, chemical state, DFS, WFS, ion flux, permeability, xylem cell walls, tomato, bromium, nitrate     

Evolutionary stability of the R1 retrotransposable element in the genus Drosophila

R1 is a non-long terminal repeat (non-LTR) retrotransposable element that inserts into a specific sequence of insect 28S ribosomal RNA genes. We have previously shown that this element has been maintained through vertical transmission in the melanogaster species subgroup of Drosophila. To address whether R1 elements have been vertically transmitted for longer periods of evolutionary time, the analysis has been extended to 11 other species from four species groups of the genus Drosophila (melanogaster, obscura, testecea, and repleta). All sequenced elements appeared functional on the basis of the preservation of their open-reading frames and consistently higher rate of substitution at synonymous sites relative to replacement sites. The phylogenetic relationships of the R1 elements from all species analyzed were congruent with the species phylogenies, suggesting that the R1 elements have been vertically transmitted since the inception of the Drosophila genus, an estimated 50-70 Mya. The stable maintenance of R1 through the germ line appears to be the major mechanism for the widespread distribution of these elements in Drosophila. In two species, D. neotestecea of the testecea group and D. takahashii of the melanogaster group, a second family of R1 elements was also present that differed in sequence by 46% and 31%, respectively, from the family that was congruent with the species phylogeny. These second families may represent occasional horizontal transfers or, alternatively, they could reflect the ability of R1 elements to diverge into new families within a species and evolve independently.      

Morphological study of bacteria of the respiratory system using fluorescence microscopy of Papanicolaou-stained smears with special regard to the identification of Mycobacteria sp.

In Papanicolaou-stained smears certain structures such as nucleoli, Pneumocystis carinii , Charcot-Leyden crystals, bacteria and fungi show a brilliant fluorescence. the morphological characteristics of microorganisms which can be detected by this system, especially mycobacteria, are described. This screening method offers the possibility of providing the clinician with a provisional diagnosis within hours. Proof of the nature of the organisms should be obtained by culture.     

Cultivation and Differentiation of Dissociated Cells of Chick Embryo Encephalon

Dissociated cells from cerebral hemispheres of chick embryo at stages 17–18, 12–13 and 9–10, were cultivated for seven days. The cells were cultivated either completely covered with the nutrient medium in an atmosphere containing 5 percent CO₂ or they were covered by only a thin film of nutrient medium in contact with air.
For the embryos at stages 17–18 or 12–13, under both culture conditions neurons differentiated after 3 or 4 days in culture, while for the embryos at stage 9–10, no neuronal differentiation occurred under either condition. The cells remained morphologically undifferentiated and formed aggregates of about 50 cells. Some fibroblasts were found to grow on the collagen matrix.
It is concluded that dissociated cells from embryos at stage 9–10 are incapable of auto differentiation under the present culture conditions. The possible causes of this inability to differentiate are discussed.     

Sequence-dependent modulation of frameshift mutagenesis at NarI-derived mutation hot spots.

The NarI sequence is known to be the strongest mutation hot spot for induced frameshift mutagenesis. Indeed, a single N-2-acetylaminofluorene (AAF) adduct induces -2 frameshift mutations (5'-GGCGAAFCC--> 5'-GGCC) more than 10(7)-fold over background mutagenesis in Escherichia coli. The mechanism of induction of the frameshift mutation involves a two nucleotide primer-template misalignment event during replication of the adduct-containing sequence. The slipped mutagenic intermediate (SMI) that is thus formed is strongly stabilised by the AAF residue. In order to understand the origin of the extreme susceptibility of this sequence to frameshift mutagenesis, we analysed AAF-induced mutagenesis at sequences 5'-NaGCGAAFCNb-3' containing the core dinucleotide GCGC repeat present in the NarI sequence flanked by variable nucleotides Na and Nb. The nature of nucleotide Nb was found to strongly modulate the frequency of induced -2 frameshift mutagenesis (up to 30 to 50-fold), while little if any effect could be attributed to nucleotide Na. The induction of -2 frameshifts, regardless of nucleotides Na and Nb, was found to be SOS-inducible but umuDC-independent as previously found for the authentic NarI sequence. The NarI sequence (GGCGCC) and sequence TGCGCA (Na=T, Nb=A) were found to be equally "hot" for -2 frameshift mutation induction compared to the sequence AGCGCT where induced mutagenesis was 30 to 50-fold lower.The analysis of replication events using constructions containing a strand marker across from the adduct site allowed us to demonstrate that the large difference in -2 frameshift mutagenesis is due to an intrinsic difference in the propensity of these sequences to slip during replication. How the nature of the nucleotide flanking the adduct on its 3'-side (Nb) differentially stabilises the SMI will be discussed in the light of recent structural data and theoretical models.     

Analysis of a genetic hitchhiking model, and its application to DNA polymorphism data from Drosophila melanogaster

Begun and Aquadro have demonstrated that levels of nucleotide variation correlate with recombination rate among 20 gene regions from across the genome of Drosophila melanogaster. It has been suggested that this correlation results from genetic hitchhiking associated with the fixation of strongly selected mutants. The hitchhiking process can be described as a series of two-step events. The first step consists of a strongly selected substitution wiping out linked variation in a population; this is followed by a recovery period in which polymorphism can build up via neutral mutations and random genetic drift. Genetic hitchhiking has previously been modeled as a steady-state process driven by recurring selected substitutions. We show here that the characteristic parameter of this steady-state model is alpha v, the product of selection intensity (alpha = 2Ns) and the frequency of beneficial mutations v (where N is population size and s is the selective advantage of the favored allele). We also demonstrate that the steady-state model describes the hitchhiking process adequately, unless the recombination rate is very low. To estimate alpha v, we use the data of DNA sequence variation from 17 D. melanogaster loci from regions of intermediate to high recombination rates. We find that alpha v is likely to be > 1.3 x 10(-8). Additional data are needed to estimate this parameter more precisely. The estimation of alpha v is important, as this parameter determines the shape of the frequency distribution of strongly selected substitutions.