Disrupted phylogeographical microsatellite and chloroplast DNA patterns indicate a vicariance rather than long‐distance dispersal origin for the disjunct distribution of the Chilean endemic Dioscorea biloba (Dioscoreaceae) around the Atacama Desert

Aim The Chilean endemic Dioscorea biloba (Dioscoreaceae) is a dioecious geophyte that shows a remarkable 600 km north–south disjunction in the peripheral arid area of the Atacama Desert. Its restricted present‐day distribution and probable Neogene origin indicate that its populations have a history linked to that of the Atacama Desert, making this an ideal model species with which to investigate the biogeography of the region. Location Chile, Atacama Desert and peripheral arid area. Methods Two hundred and seventy‐five individuals from nine populations were genotyped for seven nuclear microsatellite loci, and plastid trnL–F and trnT–L sequences were obtained for a representative subset of these. Analyses included the estimation of genetic diversity and population structure through clustering, Bayesian and analysis of molecular variance analyses, and statistical parsimony networks of chloroplast haplotypes. Isolation by distance was tested against alternative dispersal hypotheses. Results Microsatellite markers revealed moderate to high levels of genetic diversity within populations, with those from the southern Limarí Valley showing the highest values and northern populations showing less exclusive alleles. Bayesian analysis of microsatellite data identified three genetic groups that corresponded to geographical ranges. Chloroplast phylogeography revealed no haplotypes shared between northern and southern ranges, and little haplotype sharing between the two neighbouring southern valleys. Dispersal models suggested the presence of extinct hypothetical populations between the southern and northern ranges. Main conclusions Our results are consistent with prolonged isolation of the northern and southern groups, mediated by the life‐history traits of the species. Significant isolation was revealed at both large and moderate distances as gene flow was not evident even between neighbouring valleys. Bayesian analyses of microsatellite and chloroplast haplotype diversity identified the southern area of Limarí as the probable area of origin of the species. Our data do not support recent dispersal of D. biloba from the southern range into Antofagasta, but indicate the fragmentation of an earlier wider range, concomitant with the Pliocene–Pleistocene climatic oscillations, with subsequent extinctions of the Atacama Desert populations and the divergence of the peripheral ones as a consequence of genetic drift.     

Characterisation of Trypanosoma cruzi populations by dna polymorphism of the cruzipain gene detected by single-stranded dna conformation polymorphism (SSCP) and direct sequencing

Fifty fresh isolates of Trypanosoma cruzi from Triatoma dimidiata vectors and 31 from patients with Chagas disease were analysed for DNA polymorphisms within the 432-bp core region of the cruzipain gene which encodes the active site of cathepsin L-like cystein proteinase. The cruzipain gene showed signs of polymorphism consisting of four different DNA sequences in Central and South American isolates of T. cruzi. The PCR fragments of Guatemalan isolates could be divided into three groups, Groups 1, 2 and 3, based on different patterns of single-stranded DNA conformation polymorphism. All of the strains isolated from Brazil, Chile, and Paraguay, except for the CL strain, showed a Group 4 pattern. Two to four isolates from each group were analysed by cloning and sequencing. A silent mutation occurred between Groups 1 and 2, and five nucleotides and two aa substitutions were detected between Groups 1 and 3. The DNA sequence of Group 4 contained five nucleotides and one aa substitution from Group 1. All of the DNA sequences corresponded well with the single-stranded DNA conformation polymorphism. The Group 1 isolates, the majority in the Guatemalan population (70/81, 86.4%), were isolated from both triatomines and humans, but Group 3 were isolated only from humans. Moreover, the Group 2 isolates were detected only in triatomine vectors (9/50; 18%), but never in humans (0/32, P<0.05) suggesting that this group has an independent life-cycle in sylvatic animals and is maintained by reservoir hosts other than humans.     

Promotion by phloroglucinol of micropropagation of Minuartia valentina, an endangered and endemic Spanish plant

Tissue culture techniques for the propagation and conservation of endemic or threatened plants can be used to complement the methods usually applied in ex situ conservation. Thus, Minuartia valentina (Caryophyllaceae), an endangered plant species endemic to the Valencia Community (Eastern Spain), was successfully regenerated through shoot proliferation from wild plants growing in their natural area. Nodal segments, 10~mm long, were cut from rametes of adult material, sterilised and established in vitro. Equally successful shoot multiplication was achieved on Murashige and Skoog (MS) medium with 80 mg l-1 phloroglucinol in combination with either 1 mg l-1 6-benzylaminopurine or 1 mg l-1 kinetin. Excised shoots were rooted in MS medium supplemented with an auxin (indole acetic acid, indole-3-butyric acid, or napththalene acetic acid). Shoots rooted well (96–100%) within three weeks in all auxin treatments. However, the use of napththalene acetic acid was discarded because this auxin delayed root differentiation, and induced adventitious root malformation. Rooted plantlets were transferred to pots and 85% of them acclimatized successfully four weeks after transfer to greenhouse conditions, where they exhibited normal morphology and growth.     

Effect of mixing on biogas production during mesophilic anaerobic digestion of screened dairy manure in a pilot plant

The effect of mixing on biogas production of a 1.5‐m³ pilot continuous stirred tank reactor (CSTR) processing screened dairy manure was evaluated. Mixing was carried out by recirculation of reactor content with a mono pump. The experiment was conducted at a controlled temperature of 37±1°C and hydraulic retention times (HRTs) of 20 and 10 days. The effect of continuous and intermittent operation of the recirculation pump on biogas production was studied. At 10 days of HRT, the results showed a minimal influence of recirculation rate on biogas production and that continuous recirculation did not improve reactor performance. At 20 days of HRT, the recirculation rate did not affect reactor performance. Combination of low solid content in feed animal slurry and long HRTs results in minimal mixing requirements for anaerobic digestion.     

Effect of organic and conventional farming on soil bacterial diversity of pecan tree (Carya illinoensis K. Kosh) orchard across two phenological stages

We described the bacterial diversity of walnut grove soils under organic and conventional farming. The bacterial communities of rhizospheric and nonrhizospheric soils of pecan tree (Carya illinoensis K. Koch) were compared considering two phenological stages (sprouting and ripening). Sixteen operational taxonomic units (OTUs) were identified significantly more abundant according to the plant development, only one according to the farming condition, and none according to the soil origin. The OTUs specificaly abundant according to plant development included Actinobateria (2) and Betaproteobacteria (1) related OTUs more abundant at the sprouting stage, while at the fruit ripening (FR) stage the more abundant OTUs were related to Actinobacteria (6), Alphaproteobacteria (6), and unclassified Bacteria (1). The Gaiellaceae OTU18 (Actinobacteria) was more abundant under conventional farming. Thus, our study revealed that the plant development stage was the main factor shaping the bacterial community structure, while less influence was noticed for the farming condition. The bacterial communities exhibited specific metabolic capacities, a large range of carbon sources being used at the FR stage. The identified OTUs specifically more abundant represent indicators providing useful information on soil condition, potential tools for the management of soil bacterial communities.     

Transmission electron microscopic studies on the mitotic cycle of nucleolar proteins impregnated with silver

Hela cells were impregnated with silver according to Paweletz et al. (1967). In cells in mitosis not only the nucleolar organizer regions (NORs) are strongly impregnated but also part of the nucleolar material, which accumulates in and around the chromosomes. The treatment with adenosine, which in interphase cells spreads the nucleolar material within the nucleus, also distributes the argentophilic material in and around the chromosomes. During the reconstruction phase this material reassembles around the NORs to form parts of the new nucleolus. The silver impregnation technique clearly demonstrates that two main components are responsible for the argentophily of the nucleolus. This is in agreement with the results obtained by Lischwe et al. (1979).