Localization and neuroanatomy of catecholaminergic neurons in some rotifer species

We studied Dicranophorus sp., Platyias quadricornis (Ehrb.) and Rotaria tardigrada (Ehrb.). These rotifers, systematically distant from each other, show the same pattern of the catecholaminergic (CA-ergic) part of the nervous system. It is formed of a small (23–24), but steady number of neurons characteristic for each species. Three types of CA-ergic neurons are described. The sizes of neurons vary from to two to ten µm. The distribution of the brain neurons is correlated with body shape. Such a type of nervous system is topographically comparable to the concentrated orthogon of the flatworms.     

Phylogenetic position of Dictyostelium inferred from multiple protein data sets

The phylogenetic position of Dictyostelium inferred from 18S rRNA data contradicts that from protein data. Protein trees always show the close affinity of Dictyostelium with animals, fungi, and plants, whereas in 18S rRNA trees the branching of Dictyostelium is placed at a position before the massive radiation of protist groups including the divergence of the three kingdoms. To settle this controversial issue and to determine the correct position of Dictyostelium, we inferred the phylogenetic relationship among Dictyostelium and the three kingdoms Animalia, Fungi, and Plantae by a maximum-likelihood method using 19 different protein data sets. It was shown at the significance level of 1 SE that the branching of Dictyostelium antedates the divergence of Animalia and Fungi, and Plantae is an outgroup of the Animalia-Fungi-Dictyostelium clade.Correspondence to: T. Miyata     

Evolutionary analysis of the picornavirus family

An exhaustive evolutionary analysis of the picornavirus family has been carried out using the amino acid sequences of several proteins of the viruses including: the capsid proteins (1D, 1B, and 1C) situated at the 5 end of the genome and responsible for the serotype of the viruses, and the viral polymerase (3D), located at the 3 end of the genome. The evolutionary relationships found among the viruses studied support the new classification, recently suggested, in contrast to the classical one, and the existence of a new genus for the picornavirus family. In the new taxonomic organization, five genera form the picornavirus family: (1) aphthoviruses, (2) cardioviruses, (3) hepatoviruses (previously classified as enteroviruses), (4) renteroviruses (which mainly constitute a combination of the previous genera rhinovirus and enterovirus), and (5) a new genus, with a new and unique representative: the echovirus 22. Our analysis also allowed us, for the first time, to propose the most probable sequence of speciation events to have given rise to the current picornavirus family.The bootstrap procedure was used to check the reliability of the phylogenetic trees obtained. The application of the method of the statistical geometry in distance space to internal branches of the tree revealed a high degree of evolutionary noise, which makes the resolution of some internal branching points difficult. Correspondence to: J. Dopazo     

A method for determining the position and size of optimal sequence regions for phylogenetic analysis

The availability of fast and accurate sequencing procedures along with the use of PCR has led to a proliferation of studies of variability at the molecular level in populations. Nevertheless, it is often impractical to examine long genomic stretches and a large number of individuals at the same time. In order to optimize this kind of study, we suggest a heuristic procedure for detection of the shortest region whose informational content can be considered sufficient for significant phylogenetic reconstruction. The method is based on the comparison of the pairwise genetic distances obtained from a set of sequences of reference to those obtained for different windows of variable size and position by means of a simple index. We also present an approach for testing whether the informative content in the stretches selected in this way is significantly different from the corresponding content shown by the larger genomic regions used as reference. Application of this test to the analysis of the VP1 protein gene of foot-and-mouth-disease type C virus allowed us to define optimal stretches whose informative content is not significantly different from that displayed by the complete VP1 sequence. We showed that the predictions made for type C sequences are valid for type O sequences, indicating that the results of the procedure are consistent. Correspondence to: J. Dopazo     

New methods for estimating the numbers of synonymous and nonsynonymous substitutions

New methods for estimating the numbers of synonymous and nonsynonymous substitutions per site were developed. The methods are unweighted pathway methods based on Kimura's two-parameter model. Computer simulations were conducted to evaluate the accuracies of the new methods, Nei and Gojobori's (NG) method, Miyata and Yasunaga's (MY) method, Li, Wu, and Luo's (LWL) method, and Pamilo, Bianchi, and Li's (PBL) method. The following results were obtained: (1) The NG, MY, and LWL methods give overestimates of the number of synonymous substitutions and underestimates of the number of nonsynonymous substitutions. The major cause for the biased estimation is that these three methods underestimate the number of synonymous sites and overestimate the number of nonsynonymous sites. (2) The PBL method gives better estimates of the numbers of synonymous and nonsynonymous substitutions than those obtained by the NG, MY, and LWL methods. (3) The new methods also give better estimates of the numbers of synonymous and nonsynonymous substitutions than those obtained by the NG, MY, and LWL methods. In addition, estimates of the numbers of synonymous and nonsynonymous sites obtained by the new methods are reasonably accurate. (4) In some cases, the new methods and the PBL method give biased estimates of substitution numbers. However, from the number of nucleotide substitutions at the third position of codons, we can examine whether estimates obtained by the new methods are good or not, whereas we cannot make an examination of estimates obtained by the PBL method. (5) When there are strong transition/transversion and nucleotide-frequency biases like mitochondrial genes, all of the above methods give biased estimates of substitution numbers. In such cases, Kondo et al.'s method is recommended to be used for estimating the number of synonymous substitutions, although their method cannot estimate the number of nonsynonymous substitutions and is time-consuming. These results, particularly result (1), call for reexaminations of some genes. This is because evolutionary pictures of genes have often been discussed on the basis of results obtained by the NG, MY, and LWL methods, which are favorable for the neutral theory of molecular evolution.     

Distribution and production ofChironomus salinarius (Diptera: Chironomidae) in a shallow coastal lagoon in the Bay of Cádiz

The abundance, generation time and production ofChironomus salinarius larvae in a lagoon fish-pond system in the Bay of Cádiz were studied by taking monthly samples at 3 sites during 1991 and 1992. Numerical abundance and biomass of larvae showed considerable spatial, seasonal and interannual variation (ANCOVAs,P<0.001). The maximum mean annual density was 7048 larvae m^–2, and corresponded to a biomass of 3.08 g dry weight (DW) m^–2. It was recorded at the site with the lowest rate of water renewal. Seasonal patterns were similar at all sites, with main annual peaks of abundance and biomass in autumn-early winter. Chironomid density was positively related to the biomass of benthic macroalgae (P<0.001). The population studied was multivoltine with a probable average of five generations per year, with overlapping cohorts and a predominance of third- and fourth-instar larvae. Estimates of annual production ranged between 72.2 g DW m^–2 yr^–1 at the site with the lowest rate of water renewal in 1991 and 0.1 g DW m^–2 yr^–1 at the site with the highest rate of water renewal in 1992. Mean annual production and the production/biomass ratio for the system was estimated to be 16.8 g DW m^–2 yr^–1 and 12.7, respectively. Possible factors leading to the observed density fluctuations are discussed, as well as possible sources of error in production estimates.     

Sexual differences in the Golgi apparatus of rat hepatocytes: three-dimensional analysis

Lipid metabolism takes place in the Golgi apparatus, but at a higher rate in female than in male rats. I therefore examined the Golgi apparatus by morphometric means for differences between the sexes at the light-and electron-microscopic level. The Golgi apparatus was stained in situ by a zinc-iodide-osmium method. The counts of the Golgi apparatus in cross-sections in female hepatocytes by light microscopy were approximately twice that in male hepatocytes. Upon ovariectomy, these counts were greatly reduced but were reestablished after estrogen supplement. To clarify this phenomenon, three-dimensional reconstructions of the Golgi apparatus were produced from electron-microscopic images of serially cut 160-nm sections. The Golgi apparatus of both male and ovariectomized females had the shape of a small ring, whereas it took the form of a large elongated cylinder in normal females and in castrated males after treatment with estrogen. The numerical difference in Golgi apparatus counts by light microscopy of in males and females is, therefore, apparently attributable to the size and shape of the Golgi apparatus, and is controlled by the estrogen level.     

Standardized extraction method for paralytic shellfish toxins in phytoplankton

The optimal conditions were established for extraction of paralytic shellfish toxins from a Danish clone of Alexandrium tamarense using extraction with acetic acid and HCl in the concentration range 0.01–1.0 N. Physical destruction of the cells was investigated microscopically to select the most efficient extraction procedure.The toxin content was quantitated by an automized isocratic reversed-phase high-performance liquid chromatography (HPLC) method. The best results as judged from the total amount of toxins and the toxin profile were obtained using 0.05–1.0 N acetic acid and 0.01–0.02 N HCl. Hydrochloric acid in the concentration range 0.03–1.0 N caused the amount of C1 and C2 toxins to decrease sharply and concomitant increase of gonyautoxins 2 and 3.The phytoplankton extracts with 0.1 to 0.5 N acetic acid or 0.01 N HCl were stable during 6 months at –20 °C, but the extracts with HCl 0.02 N underwent a change in toxin profile, although the total amount of toxins was constant.     

HPLC-analysis of algal pigments: comparison of columns,column properties and eluents

The effects of columns (Nucleosil C₁₈ODS, MZ-PAH, YMC-PACK C₃₀), column properties (inner diameters of 4 mm, 3 mm and 2 mm, pore-width 10 nm and 30 nm) and eluents (methanol, acetonitrile, acetone, water) were tested on the separation of algal pigments. The length of columns was 250 mm and particle size was 5 μm. Flow rates and gradients were adjusted to optimize peak separation; remaining chromatographic conditions were kept constant. The resolution of chromatographic systems was tested with pigment standards and various algal cultures. Total flow rate and retention times decreased with decreasing inner diameter, whereas pressure, sensitivity and peak-width increased. Pore width had negligible effects on the chromatographic separation of pigments under the test conditions. Only with acetonitrile as eluent were all the taxonomically important pigments resolved adequately: zeaxanthin (Cyanophyceae), lutein (Chlorophyceae), fucoxanthin (Bacillariopyceae), alloxanthin (Cryptophyceae), peridinin (Dinophyceae).     

Development of needle retention in Scots pine (Pinus sylvestris) in 1957–1991 in northern and southern Finland

The needle trace method was used to study retrospectively the long-term latitudinal variation in needle retention in Scots pine (Pinus sylvestris L.) in Finland. The mean annual summer needle retention (ANR) along the main stem varied from 3.4 to 6.0 needle sets during the period 1957–1991. The lowest values were observed in southern and the highest in northern Finland. The length of the growing season, expressed as the thermal sum (threshold value +5 °C), was negatively correlated with the mean ANR (r=-0.96). The geographical needle retention pattern (NRP) for the period 1957–1991 showed a clearly increasing trend from 1957 to 1969 (southern Finland) and to 1975 (northern Finland); thereafter, the NRP tended to decrease close to its minimum value recorded in 1991. The general level of the NRP was approximately 5.0 needle sets in northern Finland and 3.5–4.0 needle sets in southern Finland. The NRP, with its 6–12 year cycle for southern Finland, was clearly periodical. Differences in the NRP among the ten stands in southern Finland were small, whereas the said periodicity was missing and the differences were high among the stands in northern Finland. The results indicate that variation in the number of needle sets, viz. defoliation of pines, is a normal phenomenon. The role of net carbon assimilation as a regulator of the number of needle sets is discussed.