Phylogeography of the cactophilic species Drosophila gouveai: demographic events and divergence timing in dry vegetation enclaves in eastern Brazil

Aim  The aim of this study was to assess the causal mechanisms underlying populational subdivision in Drosophila gouveai , a cactophilic species associated with xeric vegetation enclaves in eastern Brazil. A secondary aim was to investigate the genetic effects of Pleistocene climatic fluctuations on these environments.
Location  Dry vegetation enclaves within the limits of the Cerrado domain in eastern Brazil.
Methods  We determined the mitochondrial DNA haplotypes of 55 individuals (representing 12 populations) based on sequence data of a 483-bp fragment from the cytochrome c oxidase subunit II (COII) gene. Phylogenetic and coalescent analyses were used to test for the occurrence of demographic events and to infer the time of divergence amongst genetically independent groups.
Results  Our analyses revealed the existence of two divergent subclades (G1 and G2) plus an introgressed clade restricted to the southernmost range of D. gouveai . Subclades G1 and G2 displayed genetic footprints of range expansion and segregated geographical distributions in south-eastern and some central highland regions, east and west of the Paraná River valley. Molecular dating indicated that the main demographic and diversification events occurred in the late to middle Pleistocene.
Main conclusions  The phylogeographical and genetic patterns observed for D. gouveai in this study are consistent with changes in the distribution of dry vegetation in eastern Brazil. All of the estimates obtained by molecular dating indicate that range expansion and isolation pre-dated the Last Glacial Maximum, occurring during the late to middle Pleistocene, and were probably triggered by climatic changes during the Pleistocene. The current patchy geographical distribution and population subdivision in D. gouveai is apparently closely linked to these past events.     

Culture of chondrocytes in medium supplemented with fetal calf serum or a serum substitute: Ultroser G

Fetal calf serum and a serum substitute, Ultroser G, were compared for their effects on the growth curves, clonal growth and cell cycle progression of rabbit chondrocytes in primary culture and during at least three cell passages and included a screen for the maintenance of cartilage-like differentiation i.e. the presence of type II collagen. Proliferation was also compared with another serum substitute, Nu-Serum. Ultroser G is shown to be equivalent to fetal calf serum as far as chondrocyte proliferation is concerned, clonal growth is improved and biosynthesis of type II collagen is maintained in primary culture.     

Liposome-mediated DNA transfer in eukaryotic cells: Dependence of the transfer efficiency upon the type of liposomes used and the host cell cycle stage

The pBR322 plasmid containing the sequence encoding β-lactamase, the enzyme conferring resistance to ampicillin, was encapsulated in liposomes of different phospholipid composition and incubated with synchronized cells. In mitotic cells as compared to cells synchronized in G₁, twice as many exogeneous DNA molecules were found associated with the cell nuclear DNA, when fluid, neutral liposomes were used. These liposomes are taken up by the cells mainly via endocytosis. When fluid, negatively charged liposomes were used as carriers about the same number of exogeneous DNA molecules were found associated with the nuclear DNA both in mitotic and in G₁-synchronized cells. The efficiency for gene transfer of liposomes entering the cells by different mechanisms was further studied and expressed both by the fraction of the radioactive plasmid associated with the nuclear DNA and by the level of the β-lactamase activity detected in the transfected cells. It appears that liposomes entering the cells mainly via an energy-dependent mechanism are more efficient for this type of DNA transfer.     

Properties of succinylated wheat-germ agglutinin.

The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a pI of 4.0 +/- 0.2 while the native lectin is basic, pI of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N-acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 microgram/ml with both lectins and the concentrations of N-acetylglucosamine and di-N-acetylchitobiose which inhibit agglutination are similar with both lectins. The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins. The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface.     

Non-native prey species supporting fish assemblage biomass in a Neotropical reservoir

The present study aimed to investigate the role of four non-native invertebrates in supporting fish biomass as well as their influence on the carbon flow into the Volta Grande reservoir food web. The fish samples were carried out quarterly between October 2015 and July 2016 using gillnets. At the sampled sites, four non-native invertebrates (golden mussel, Asian clam, trumpet snail and Amazonian prawn), which are potential prey for fish in the Volta Grande reservoir, were collected by targeted sampling using a Petersen-type bottom dredger and semi-circular sieves. The gut contents of the fish were collected and analyzed under stereoscope, and samples of muscle tissue of these fish, as well as the four non-native invertebrate species sampled, were submitted for isotopic analysis. Results obtained by the present study, by both gut content and stable isotopic analyses, pointed to a trophic structure where non-native species represent not only a strong component of the fish community, but also their main carbon source. Based on gut contents and isotopic mixing models, we found that together, non-native prey are essential carbon sources for the fish fauna, fuelling more than 40.0% of the biomass in four dominant fish species. The consumption rate of non-native bivalves by the native omnivorous fish Leporinus friderici suggested these filter-feeding organisms potentially constitute an important trophic connection between littoral consumers and pelagic energy sources. In addition, non-native prey were also prominent carbon sources for non-native fish, fuelling more than half of the biomass in peacock bass and silver croaker, suggesting these prey have a fundamental role in maintaining non-native fish populations in this system. Our results may help to understand fundamental ecological issues bringing to light the extent to which these new combinations of species influence the energy flow and ecosystem properties of the Volta Grande reservoir.

     

Low courtship song variation in south and southeastern Brazilian populations of Drosophila meridionalis (Diptera, Drosophilidae)

Drosophila meridionalis is a cactus-breeding species with a wide distribution in South America. Most populations of this species are geographically isolated, what provides a promising scenario for studying evolution. Former studies of this species revealed a remarkable karyotypic variation among its populations. Up to six distinct metaphase chromosomes were described, showing that this species is polymorphic at least at the chromosomal level. In order to elucidate the taxonomic status of populations showing different metaphase chromosomes, we analyzed the courtship song of five populations of D. meridionalis in South and Southeastern Brazil. In addition, we analyzed the metaphase chromosomes of each population. Our results show that, despite the two karyotype observed, most courtship song parameters did not vary among the populations. Altogether, our results suggest that D. meridionalis from South and Southeastern Brazil represents one species with an inter-population chromosomal variability.     

Allozymatic divergence between border populations of two cryptic species of the Drosophila buzzatii cluster species (Diptera: Drosophilidae)

Drosophila antonietae and Drosophila gouveai are allopatric, cactophilic, cryptic and endemic of South America species, which aedeagus morphology is considered the main diagnostic character. In this work, single close populations from the edge distributions of each species, located in an “introgressive corridor”, were analyzed regarding temporal isozenzymatic genetic variability. Isocitrate dehydrogenase (Idh) appeared as a diagnostic locus between D. antonieate and D. gouveai because each population was fixed for different alleles. Moreover, several polymorphic loci showed accentuated divergence in the allele frequency, as evidenced by Nei’s I (0.3188) and D (1.1432), and also by Reynolds’ genetic distance and identity (1.3207 and 0.7331, respectively). Our results showed that, in spite of the very similar external morphology, related evolutionary histories, close distributions, and events of introgression in the studied area, these cryptic species have high allozymatic differentiation, and this is discussed here.     

Conservation of pBuM–2 satellite DNA sequences among geographically isolated Drosophila gouveai populations from Brazil

In this study, we have compared 34 repetition units of pBuM-2 satellite DNA of individuals from six isolated populations of Drosophila gouveai, a cactophilic member of Drosophila buzzatii cluster (repleta group). In contrast to the results of previous morphological and molecular data, which suggest differentiation among the D. gouveai populations, the sequences and the cluster analysis of pBuM-2 monomers showed that this repetitive element is highly conserved among the six D. gouveai populations (97.8% similarity), indicating a slow rate of evolution of pBuM-2 sequences at the population level. Probably, some homogenization mechanisms of tandem sequences, such as unequal crossing or gene conversion, have maintained the sequence similarity of pBuM-2 among D. gouveai populations. Alternatively, such a result may be associated with a functional role of pBuM-2 sequences, although it is not understood at present.     

Chromosome variability in Brazilian populations of Drosophila buzzatii (Diptera, Drosophilidae).

Analysis of 28 strains of Drosophila buzzatii from six different localities revealed that populations from southern Brazil apparently differ from northeastern populations in chromosome structure. Of the 16 polymorphic chromosome inversions detected in this species, only two (2j and 5c2) were present in southern populations. The Northeastern populations were the first of this species found not to have the 2j inversion. This suggests that the populations of Drosophila buzzatii may have lost chromosome polymorphism during dispersion, supporting the idea that the current geographic distribution of this species in South America is due to active dispersion.