A preliminary revision of the genus Plecotus (Chiroptera, Vespertilionidae) based on genetic and morphological results

The phylogenetic relationships within the genus Plecotus were assessed using molecular as well as morphological methods. With only three species missing, our study is based on an almost comprehensive taxonomic sampling. The genetic analysis comprised 151 individuals from throughout the range. Sequences of two mitochondrial sections, parts of the 16S rRNA gene (16S) and of the control region (CR) were analysed. The morphological analysis of cranial and external characters comprised 697 individuals, including 10 holotypes and one lectotype. Data from 15 craniometric characters of 442 specimens were used in the multivariate analyses. The molecular data identified nine primary clades representing 11 species, 10 of which could be assigned to described taxa, whereas one was described as a new species, Plecotus strelkovi Spitzenberger sp. nov. The tree based on 16S revealed two major lineages, one consisting of only one primary clade restricted to the Mediterranean, the other consisting of eight primary clades representing Eurasian taxa. The morphological analysis revealed five additional species, two of them not described. Together with the recently described P. taivanus, P. sardus and P. balensis, which were not included in our analysis, the genus Plecotus comprises at least 19 more or less cryptic species. Phylogenetic and phenetic analyses resulted in similar but not completely concordant arrangements of the species. The proposed classification relies mainly on the tree based on 16S sequences. The current distribution indicates that 16 species can be linked to arboreal refugia, three to eremial refugia. We assume that speciation within the gleaning, rather slow flying long‐eared bats is due to a multitude of disruption and isolation processes within a formerly continuous range of the broad‐leaved Arcto‐Tertiary forest in which Plecotus probably originated. An exact calibrated molecular dating of the splits is not possible. The Early Oligocene age of the presumed ancestor of the Plecotini and a correlation of the molecular diversifications with palaeogeographic reconstructions suggest that the divergence of the two major lineages may have occurred already during the Middle Miocene, 14.5 Mya.     

Tomato linalool synthase is induced in trichomes by jasmonic acid

Tomato (Lycopersicon esculentum) plants emit a blend of volatile organic compounds, which mainly consists of terpenes. Upon herbivory or wounding, the emission of several terpenes increases. We have identified and characterized the first two tomato monoterpene synthases, LeMTS1 and LeMTS2. Although these proteins were highly homologous, recombinant LeMTS1 protein produced (R)-linalool from geranyl diphosphate (GPP) and (E)-nerolidol from farnesyl diphosphate (FPP), while recombinant LeMTS2 produced β-phellandrene, β-myrcene, and sabinene from GPP. In addition, these genes were expressed in different tissues: LeMTS1 was expressed in flowers, young leaves, stems, and petioles, while LeMTS2 was strongest expressed in stems and roots. LeMTS1 expression in leaves was induced by spider mite-infestation, wounding and jasmonic acid (JA)-treatment, while LeMTS2 did not respond to these stimuli. The expression of LeMTS1 in stems and petioles was predominantly detected in trichomes and could be induced by JA. Because JA treatment strongly induced emission of linalool and overexpression of LeMTS1 in tomato resulted in increased production of linalool, we propose that LeMTS1 is a genuine linalool synthase. Our results underline the importance of trichomes in JA-induced terpene emission in tomato.     

Regulation of glucose carrier activity by AlCl3 and phospholipase C in fat-cells.

Recently it was speculated that activation of GTP-binding proteins and of phospholipase is involved in the transmission of a signal from the insulin-receptor kinase to effector systems in the cell. To confirm this hypothesis, we have tested the effect of AlCl3, which has been recently used as an experimental tool to activate GTP-binding proteins, on glucose transport in fat-cells. We found that AlCl3 has a partial insulin-like effect on glucose transport activity (3-O-methylglucose uptake, expressed as % of equilibrium value per 4 s: basal 9.6 +/- 2, AlCl3 29.6 +/- 4, insulin 74.0 +/- 3). The AlCl3 effect is totally blocked by pertussis toxin, whereas the insulin effect was not altered. The effect starts at [AlCl3] greater than 1 fM and reaches its maximum at 0.1 nM. Addition of phospholipase C (PLC; 50 munits/ml) also stimulated glucose transport (maximal 53.0 +/- 5%). Both substances acted faster than insulin itself (maximal values within 1 min for PLC, 2 min for AlCl3 and 5-10 min for insulin). Using the cytochalasin-B-binding assay to determine the effects of AlCl3 and PLC on the distribution of glucose carrier sites in subcellular fractions, we found that their glucose-transport-stimulating effect does not occur through an increase in glucose carrier sites in the plasma-membrane fraction. When PLC was combined with the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate), which increases glucose carrier sites in the plasma membrane, an additive effect on glucose transport was found [PLC (50 munits/ml), 53.0 +/- 5%, TPA (1 nM), 17.3 +/- 2%; PLC + TPA, 68.0 +/- 3%]. In conclusion: (1) the data show that AlCl3, probably through activation of a pertussis-toxin-inhibitable G protein, and PLC are able to modulate the intrinsic glucose carrier activity; (2) as pertussis toxin did not modify the effect of insulin, it seems unlikely that the insulin signal on glucose transport involves activation of this specific G protein.     

Characterization of Blue Light Signal Transduction Chains That Control Development and Maintenance of Sexual Competence in Chlamydomonas reinhardtii

Blue light induces the differentiation of Chlamydomonas reinhardtii pregametes to gametes. The light-induced conversion of pregametes to gametes is protein synthesis dependent and proceeds only after a lag phase. Upon incubation in the dark, gametes lost their mating ability, resulting in dark-inactivated gametes. Reillumination rapidly restored mating competence and this was shown to be independent of protein synthesis. Apparently, differentiation and maintenance of gametic competence are both regulated by light. Whether one or two light-activated signal pathways are involved was investigated using pharmacological compounds that affect signal transduction. Compounds that affected pregamete-to-gamete conversion affected the expression of a gamete-specific gene in a similar fashion. Other drugs affected only dark-inactivated gametes, suggesting that reactivating gametes requires a separate signaling pathway. Combined treatments provided evidence for the consecutive action of a phosphatase and a protein kinase C-like kinase in the light-induced reactivation process.     

Cubicle Refusal in Norwegian Dairy Herds

In order to survey the behaviour of choosing the alley area instead of a cubicle as a lying place (cubicle refusal), a questionnaire was sent to the 273 dairy farms in Norway known to keep cows in cubicle housing systems. Sixty-six percent of the farmers contacted were included in the study. The median herd size was 18 cows (range 7–118). More than 85% of the herds had sheds providing one or more cubicles per cow. The mean herd occurrence of cubicle refusal was 6%, but showed great variation (range 0–55%). Regression analysis showed a significant association between rearing heifers in slatted floor pens and an increased cubicle refusal occurrence (p = 0.02, R² = 0.05), while herd size, use of litter, or cubicle-to-animal ratio were not found to be associated with cubicle refusal. The practice of rearing heifers in slatted floor pens accounted for about one half of the observed cubicle refusal (etiologic fraction = 0.51).     

Characterization of five tomato phospholipase D cDNAs: rapid and specific expression of LePLDβ1 on elicitation with xylanase

Phospholipase D (PLD, EC 3.1.4.4.) has been implicated in a variety of plant processes, including signalling. In Arabidopsis thaliana a PLD gene family has been described and individual members classified into alpha-, beta- and gamma-classes. Here we describe a second PLD gene family in tomato (Lycopersicon esculentum) that includes three alpha- and two beta-classes. Different expression patterns in plant organs were observed for each PLD. In testing a variety of stress treatments on tomato cell suspensions, PLDbeta1 mRNA was found to rapidly and specifically accumulate in response to the fungal elicitor xylanase. The greatest increase was found 2 h after treatment with 100 microg m1(-1) xylanase (ninefold). In vivo PLD activity increased nearly threefold over a 1.5 h period of treatment. When the elicitor was injected into tomato leaves, PLDbeta1 mRNA accumulation peaked at 2 h (threefold increase), before decreasing to background levels within 72 h. Mutant, non-active xylanase was as effective as the active enzyme in eliciting a response, suggesting that xylanase itself, and not the products resulting from its activity, functioned as an elicitor. When chitotetraose was used as elicitor, no PLDbeta1 mRNA accumulation was observed, thus it is not a general response to elicitation. Together these data show that PLD genes are differentially regulated, reflecting potential differences in cellular function. The possibility that PLDbeta1 is a signalling enzyme is discussed.     

In vivo generation of pathogen-specific Th1 cells in the absence of the IFN-gamma receptor

The precise mechanisms that govern the commitment of CD4 T cells to become Th1 or Th2 cells in vivo are incompletely understood. Recent experiments demonstrate colocalization of the IFN-gammaR chains with the TCR during activation of naive CD4 T cells, suggesting that association of these molecules may be involved in determining lineage commitment. To test the role of IFN-gamma and its receptor in the generation of Th1 Ag-specific CD4 T cells, we analyzed mice after infection with Listeria monocytogenes or lymphocytic choriomeningitis virus. In the absence of IFN-gamma, Ag-specific CD4 T cells were generated in response to both these infections. In addition, IFN-gamma-producing (Th1) Ag-specific CD4 T cells were generated in mice lacking the ligand-binding chain of the IFN-gammaR (IFN-gammaR1-/-) or the signaling chain (IFN-gammaR2-/-). There was no increase in the number of IL-4-producing Ag-specific CD4 T cells, nor was there a decrease in the expression of T-bet in the absence of functional IFN-gamma signaling, indicating that the cells were committed Th1 cells. Thus, both chains of the IFN-gammaR are dispensable for the generation of Th1 Ag-specific CD4 T cells after infection in vivo.     

Dissection of the fusarium I2 gene cluster in tomato reveals six homologs and one active gene copy.

The I2 locus in tomato confers resistance to race 2 of the soil-borne fungus Fusarium oxysporum f sp lycopersici. The selective restriction fragment amplification (AFLP) positional cloning strategy was used to identify I2 in the tomato genome. A yeast artificial chromosome (YAC) clone covering approximately 750 kb encompassing the I2 locus was isolated, and the AFLP technique was used to derive tightly linked AFLP markers from this YAC clone. Genetic complementation analysis in transgenic R1 plants using a set of overlapping cosmids covering the I2 locus revealed three cosmids giving full resistance to F. o. lycopersici race 2. These cosmids shared a 7-kb DNA fragment containing an open reading frame encoding a protein with similarity to the nucleotide binding site leucine-rich repeat family of resistance genes. At the I2 locus, we identified six additional homologs that included the recently identified I2C-1 and I2C-2 genes. However, cosmids containing the I2C-1 or I2C-2 gene could not confer resistance to plants, indicating that these members are not the functional resistance genes. Alignments between the various members of the I2 gene family revealed two significant variable regions within the leucine-rich repeat region. They consisted of deletions or duplications of one or more leucine-rich repeats. We propose that one or both of these leucine-rich repeats are involved in Fusarium wilt resistance with I2 specificity.     

Range‐constrained co‐occurrence simulation reveals little niche partitioning among rock‐dwelling Montenegrina land snails (Gastropoda: Clausiliidae)

Aim

Taxon co‐occurrence analysis is commonly used in ecology, but it has not been applied to range‐wide distribution data of partly allopatric taxa because existing methods cannot differentiate between distribution‐related effects and taxon interactions. Our first aim was to develop a taxon co‐occurrence analysis method that is also capable of taking into account the effect of species ranges and can handle faunistic records from museum databases or biodiversity inventories. Our second aim was to test the independence of taxon co‐occurrences of rock‐dwelling gastropods at different taxonomic levels, with a special focus on the Clausiliidae subfamily Alopiinae, and in particular the genus Montenegrina.

Location

Balkan Peninsula in south‐eastern Europe (46N–36N, 13.5E–28E).

Methods

We introduced a taxon‐specific metric that characterizes the occurrence probability at a given location. This probability was calculated as a distance‐weighted mean of the taxon's presence and absence records at all sites. We applied corrections to account for the biases introduced by varying sampling intensity in our dataset. Then we used probabilistic null‐models to simulate taxon distributions under the null hypothesis of no taxon interactions and calculated pairwise and cumulated co‐occurrences. Independence of taxon occurrences was tested by comparing observed co‐occurrences to simulated values.

Results

We observed significantly fewer co‐occurrences among species and intra‐generic lineages of Montenegrina than expected under the assumption of no taxon interaction.

Main conclusions

Fewer than expected co‐occurrences among species and intra‐generic clades indicate that species divergence preceded niche partitioning. This suggests a primary role of non‐adaptive processes in the speciation of rock‐dwelling gastropods. The method can account for the effects of distributional constraints in range‐wide datasets, making it suitable for testing ecological, biogeographical, or evolutionary hypotheses where interactions of partly allopatric taxa are in question.