A modified two primer approach to oligonucleotide-directed in vitro mutagenesis

Using oligonucleotide-directed mutagenesis, we are trying to define the features of the protein structure that are important for the DNA and c-AMP binding by CAP from E. coli, the enzymic activity and putative DNA binding of dihydrofolate reductase of L. casei, and the functionally important regions of the self-splicing RNA of the r-RNA intron of Tetrahymena thermophila. We have used a modification of the method described by Norris et al. [1]. A mutagenic primer and an M13 universal sequencing primer are annealed simultaneously to a template from an M13 clone containing the DNA to be mutagenised and, after DNA strand extension, the fragment is cut out and recloned into either M13 or plasmid vectors. We have analysed the effect on the frequency of mutation of: the temperature used for strand extension; the class of base change attempted; the host mismatch repair system. A recently developed system for phenotypic detection of mutations in the Tetrahymena intron aided in determining mutation frequencies.     

New or interestingHyphomycetes on decaying pine litter from Czechoslovakia

Twenty three species ofHyphomycetes new to Czechoslovakia are listed with comments and illustrations. Seven are new to science—Blastophorum pini, Bloxmia bohemica, Junctospora pulchra, Linodochium formosum, Phaeostalagmus peregrinus, Sporidesmium doliiforme, Xiambola mirabilis—and of these, two are the types of new genera (Junctospora andXiambola). The new species and genera are described, illustrated and discussed. Anamorphs ofMytilidion gemmigenum Fuckel andPseudohelotium pineti (Batsch exFr.)Fuckel inTaeniolella andLinodochium, respectively are reported. Collections of these fungi from Britain are also cited. Data on morphology, ecology and distribution of those species occurring in both Britain and Czechoslovakia are compared.     

Intracellular calcium buffering capacity in isolated squid axons

Changes in ionized calcium were studied in axons isolated from living squid by measuring absorbance of the Ca binding dye Arsenazo III using multiwavelength differential absorption spectroscopy. Absorption changes measured in situ were calibrated in vitro with media of ionic composition similar to axoplasm containing CaEGTA buffers. Calcium loads of 50-2,500 μmol/kg axoplasm were induced by microinjection, by stimulation in 112 mM Ca seawater, or by soaking in choline saline with 1-10 mM Ca. Over this range of calcium loading of intact axoplasm, the ionized calcium in the axoplasm rose about 0.6 nM/μM load. Similar loading in axons preteated with carbonyl cyanide 4- trifluoromethoxyphenylhydrazone (FCCP) to inhibit the mitochondrial proton gradient increased ionized calcium by 5-7 percent of the imposed load, i.e. 93-95 percent of the calcium load was buffered by a process insensitive to FCCP. This FCCP- insensitive buffer system was not saturated by the largest calcium loads imposed, indicating a capacity of at least several millimolar. Treatment of previously loaded axons with FCCP or apyrase plus cyanide produced rises in ionized calcium which could be correlated with the extent of the load. Analysis of results indicated that, whereas only 6 percent of the endogenous calcium in fresh axons is stored in the FCCP-sensitive (presumably mitochondrial) buffer system, about 30 percent of an imposed exogenous load in the range of 50-2,500 μM is taken up by this system.     

Factors shaping communities of pyrophilous macrofungi in microhabitats destroyed by illegal campfires

Pyrophilous macrofungi (PM) are a narrowly specialised group appearing exclusively in plant communities recently destroyed by fire. Their significance has hitherto been studied only for vegetation destroyed over large areas, while in small areas of fire, i.e., microhabitats they are viewed as independent components of the community linked only to the substratum. In the present work, the following hypotheses were tested: (1) species structures of PM in microhabitats depend on the type of plant community, (2) PM form communities on a small scale which are similar in structure and function to analogous large scale communities. We studied 20 surfaces destroyed by illegal campfires in four natural plant communities: oak-hornbeam forest Tilio-Carpinetum (TC), lowland acidophilus beech forest Luzulo pilosae-Fagetum (LF), suboceanic pine forest Leucobryo-Pinetum (LP), and an initial-phase xerothermic grassland community on a transitional habitat (MH). TC and LF habitats were conspicuously more favourable for PM than LP and MH. In TC and LF fire leads to significant loss of mycorrhizae in the upper layer of leaf litter. This provides a development opportunity for ectomycorrhizal PM species which, having little competition, substitute for the destroyed fragments of mycorrhizal networks. In LP and MH fire over a small surface does not destroy more deeply located mycorrhizal associations. Another important factor for PM influencing the quality of environment is the fertility of soil: highest in TC, intermediate in LF and lowest in LP and MH. The results casts doubt on the concept that PM are only synusia linked to the substratum (burnt wood). PM growing in microhabitats constitute an important group of organisms which facilitate rapid regeneration of plant community fragments destroyed by fire.     

The evolution of antennal courtship in diplazontine parasitoid wasps (Hymenoptera,Ichneumonidae, Diplazontinae)

Background

As predicted by theory, traits associated with reproduction often evolve at a comparatively high speed. This is especially the case for courtship behaviour which plays a central role in reproductive isolation. On the other hand, courtship behavioural traits often involve morphological and behavioural adaptations in both sexes; this suggests that their evolution might be under severe constraints, for instance irreversibility of character loss. Here, we use a recently proposed method to retrieve data on a peculiar courtship behavioural trait, i.e. antennal coiling, for 56 species of diplazontine parasitoid wasps. On the basis of a well-resolved phylogeny, we reconstruct the evolutionary history of antennal coiling and associated morphological modifications to study the mode of evolution of this complex character system.

Results

Our study reveals a large variation in shape, location and ultra-structure of male-specific modifications on the antennae. As for antennal coiling, we find either single-coiling, double-coiling or the absence of coiling; each state is present in multiple genera. Using a model comparison approach, we show that the possession of antennal modifications is highly correlated with antennal coiling behaviour. Ancestral state reconstruction shows that both antennal modifications and antennal coiling are highly congruent with the molecular phylogeny, implying low levels of homoplasy and a comparatively low speed of evolution. Antennal coiling is lost on two independent occasions, and never reacquired. A zero rate of regaining antennal coiling is supported by maximum parsimony, maximum likelihood and Bayesian approaches.

Conclusions

Our study provides the first comparative evidence for a tight correlation between male-specific antennal modifications and the use of the antennae during courtship. Antennal coiling in Diplazontinae evolved at a comparatively low rate, and was never reacquired in any of the studied taxa. This suggests that the loss of antennal coiling is irreversible on the timescale examined here, and therefore that evolutionary constraints have greatly influenced the evolution of antennal courtship in this group of parasitoid wasps. Further studies are needed to ascertain whether the loss of antennal coiling is irreversible on larger timescales, and whether evolutionary constraints have influenced courtship behavioural traits in a similar way in other groups.