Intragenomic variation in ITS2 rDNA in the louse of humans, Pediculus humanus: ITS2 is not a suitable marker for population studies in this species

The two internal transcribed spacers (ITS) of ribosomal DNA are often used as markers of populations of insects. We studied the ITS2 of the head lice and body lice of humans, to determine whether this gene is a suitable marker of populations of these insects. ITS2 sequences were amplified by PCR from lice from four different countries: Australia, China, Japan and the USA. Direct cycle-sequencing of some of these PCR products gave equivocal nucleotide chromatograms. This indicated that some lice had more than one ITS2 sequence, so we cloned PCR products from these lice. Temperature gradient gel electrophoresis (TGGE) revealed that 50 of the 67 clones we screened had different nucleotide sequences. All lice had several ITS2 types, including those with unequivocal chromatograms. A phylogenetic tree of 15 different ITS2 sequences showed that the sequences from individual lice were not monophyletic. We conclude that the ITS2 is not a useful marker of populations for Pediculus humanus.

The p35 relative, p49, inhibits mammalian and Drosophila caspases including DRONC and protects against apoptosis

This study characterized the ability of a new member of the p35 family, p49, to inhibit a number of mammalian and insect caspases. p49 blocked apoptosis triggered by treatment with Fas ligand (FasL), Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or ultraviolet (UV) radiation but provided negligible protection against apoptosis induced by the chemotherapeutic drug cisplatin. The caspase cleavage site in p49 was determined, and mutation of the 131 residue of this site abolished the ability of p49 to inhibit caspases, implying that p49 inhibits caspases through an analogous suicide-substrate mechanism to p35. Unlike p35, p49 inhibited the upstream insect caspase DRONC.

Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases

A significant number of chimeric 16S rDNA sequences of diverse origin were identified in the public databases by partial treeing analysis. This suggests that chimeric sequences, representing phylogenetically novel non-existent organisms, are routinely being overlooked in molecular phylogenetic surveys despite a general awareness of PCR-generated artefacts amongst researchers.

Sequence analysis, chromosomal distribution and long-range organization show that rapid turnover of new and old pBuM satellite DNA repeats leads to different patterns of variation in seven species of the Drosophila buzzatii cluster

We aimed to study patterns of variation and factors influencing the evolutionary dynamics of a satellite DNA, pBuM, in all seven Drosophila species from the buzzatii cluster (repleta group). We analyzed 117 alpha pBuM-1 (monomer length 190 bp) and 119 composite alpha/beta (370 bp) pBuM-2 repeats and determined the chromosome location and long-range organization on DNA fibers of major sequence variants. Such combined methodologies in the study of satDNAs have been used in very few organisms. In most species, concerted evolution is linked to high copy number of pBuM repeats. Species presenting low-abundance and scattered distributed pBuM repeats did not undergo concerted evolution and maintained part of the ancestral inter-repeat variability. The alpha and alpha/beta repeats colocalized in heterochromatic regions and were distributed on multiple chromosomes, with notable differences between species. High-resolution FISH revealed array sizes of a few kilobases to over 0.7 Mb and mutual arrangements of alpha and alpha/beta repeats along the same DNA fibers, but with considerable changes in the amount of each variant across species. From sequence, chromosomal and phylogenetic data, we could infer that homogenization and amplification events involved both new and ancestral pBuM variants. Altogether, the data on the structure and organization of the pBuM satDNA give insights into genome evolution including mechanisms that contribute to concerted evolution and diversification.

Intra- and interspecific divergence in the nuclear sequences of the clock gene period in species of the Drosophila buzzatii cluster

We have measured nucleotide variation in the CLOCK/CYCLE heterodimer inhibition domain (CCID) of the clock X-linked gene period in seven species belonging to the Drosophila buzzatii cluster, namely D. buzzatii, Drosophila koepferae, Drosophila antonietae, Drosophila serido, Drosophila gouveai, Drosophila seriema and Drosophila borborema. We detected that the purifying selection is the main force driving the sequence evolution in period, in agreement with the important role of CCID in clock machinery. Our survey revealed that period provides valuable phylogenetic information that allowed to resolve phylogenetic relationships among D. gouveai, D. borborema and D. seriema, which composed a polytomic clade in preliminary studies. The analysis of patterns of intraspecific variation revealed two different lineages of period in D. koepferae, probably reflecting introgressive hybridization from D. buzzatii, in concordance with previous molecular data.

Low Satellite DNA Variability in Natural Populations of Drosophila antonietae Involved in Different Evolutionary Events

Drosophila antonietae is a cactophilic species that is found in the mesophilic forest of the Parana`-Paraguay river basin and in the dunes of the South Atlantic coast of Brazil. Although the genetic structure of the Parana`-Paraguay river basin populations has already been established, the relationship between these populations and those on the Atlantic coast is controversial. In this study, we compared 33 repetitive units of pBuM-2 satellite DNA isolated from individuals from 8 populations of D. antonietae in these geographic regions, including some populations found within a contact zone with the closely related D. serido. The pBuM-2 sequences showed low interpopulational variability. This result was interpreted as a consequence of both gene flow among the populations and unequal crossing over promoting homogenization of the tandem arrays. The results presented here, together with those of previous studies, highlight the use of pBuM-2 for solving taxonomic conflicts within the D. buzzatii species cluster.

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 l(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. (C) 2010 Elsevier Ltd. All rights reserved.

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 Parana 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.

Seasonal dynamics of a drosophilid (Diptera) assemblage and its potencial as bioindicator in open environments

Drosophila Fallen, 1823 (Diptera, Drosophilidae) is for long a well-established model organism for genetics and evolutionary research. The ecology of these flies, however, has only recently been better studied. Recent papers show that Drosophila assemblies can be used as bioindicators of forested environment degradation. In this work the bioindicator potential of drosophilids was evaluated in a naturally opened environment, a coastal strand-forest (restinga). Data from nine consecutive seasonal collections revealed strong temporal fluctuation pattern of the majority of Drosophila species groups. Drosophila willistoni group was more abundant at autumns, whereas D. cardini and D. tripunctata groups were, respectively, expressive at winters and springs, and D. repleta group at both seasons. The exotic species D. simulans Sturtevant, 1919 (from D. melanogaster group) and Zaprionus indianus Gupta, 1970 were most abundant at summers. Overall, the assemblage structure did not show the same characteristics of forested or urban environments, but was similar to the forests at winters and to cities at summers. This raises the question that this locality may already been under urbanization impact. Also, this can be interpreted as an easily invaded site for exotic species, what might lead to biotic homogenization and therefore can put in check the usage of drosophilid assemblages as bioindicators at open environments.

Genetic basis of the formation and identity of type I and type II neurons in Drosophila embryos

The embryonic peripheral nervous system of Drosophila contains two main types of sensory neurons: type I neurons, which innervate external sense organs and chordotonal organs, and type II multidendritic neurons, Here, we analyse the origin of the difference between type I and type II in the case of the neurons that depend on the proneural genes of the achaete-scute complex (ASC), We show that, in Notch(-) embryos, the type I neurons are missing while type nr neurons are produced in excess, indicating that the type I/type II choice relies on Notch-mediated cell communication, In contrast, both type I and type II neurons are absent in numb(-) embryos and after ubiquitous expression of tramtrack, indicating that the activity of numb and the absence of tramtrack are required to produce both external sense organ and multidendritic neural fates, The analysis of string(-) embryos reveals that when the precursors are unable to divide they differentiate mostly into type II neurons, indicating that the type II is the default neuronal fate, We also report a new mutant phenotype where the ASC-dependent neurons are converted into-type II neurons, providing evidence for the existence of one or more genes required for maintaining the alternative (type I) fate, Our results suggest that the same mechanism of type I/type II specification may operate at a late step of the ASC-dependent lineages, when multidendritic neurons arise as siblings of the external sense organ neurons and, at an early step, when other multidendritic neurons precursors arise as siblings of external sense organ precursors.

Leishmania sp identification by PCR associated with sequencing of target SSU rDNA in paraffin-embedded skin samples stored for more than 30 years

Paraffin-embedded samples commonly stored at educational and research institutions constitute tissues banks for follow-up or epidemiological studies; however, the paraffin inclusion process involves the use of substances that can cause DNA degradation. In this study, a PCR protocol was applied to identify Leishmania strains in 33 paraffin-embedded skin samples of patients with American cutaneous leishmaniasis. DNA was obtained by the phenol-chloroform protocol following paraffin removal and then used in PCR or nested PCR based on the nucleotide sequence of the small subunit ribosomal RNA (SSU rDNA). The amplicons obtained were cloned and sequenced to determine the single nucleotide polymorphism that distinguishes between different Leishmania species or groups. This assay allowed to distinguish organisms belonging to the subgenus Viannia and identify L. (Leishmania) amazonensis and L. (L.) chagasi of the Leishmania subgenus. Of the 33 samples, PCR and nested PCR identified 91% of samples. After sequencing the PCR product of 26 samples, 16 were identified as L. (L.) amazonensis, the other 10 contain organisms belonging to the L. (Viannia) sub-genus. These results open a huge opportunity to study stored samples and promote relevant contributions to epidemiological studies.

Phylogenetic analysis of the Monocotylidae (Monogenea) inferred from 28S rDNA sequences

The current classification of the Monocotylidae (Monogenea) is based on a phylogeny generated from morphological characters. The present study tests the morphological phylogenetic hypothesis using molecular methods. Sequences from domains C2 and D1 and the partial domains C1 and D2 from the 28S rDNA gene for 26 species of monocotylids from six of the seven subfamilies were used. Trees were generated using maximum parsimony, neighbour joining and maximum likelihood algorithms. The maximum parsimony tree, with branches showing less than 70% bootstrap support collapsed, had a topology identical to that obtained using the maximum likelihood analysis. The neighbour joining tree, with branches showing less than 70% support collapsed. differed only in its placement of Heterocotyle capricornensis as the sister group to the Decacotylinae clade. The molecular tree largely supports the subfamilies established using morphological characters. Differences are primarily how the subfamilies are related to each other. The monophyly of the Calicotylinae and Merizocotylinae and their sister group relationship is supported by high bootstrap values in all three methods, but relationships within the Merizocotylinae are unclear. Merizocotyle is paraphyletic and our data suggest that Mycteronastes and Thaumatocotyle, which were synonymized with Merizocotyle after the morphological cladistic analysis, should perhaps be resurrected as valid genera. The monophyly of the Monocotylinae and Decacotylinae is also supported by high bootstrap values. The Decacotylinae, which was considered previously to be the sister group to the Calicotylinae plus Merizocotylinae, is grouped in an unresolved polychotomy with the Monocotylinae and members of the Heterocotylinae. According to our molecular data, the Heterocotylinae is paraphyletic. Molecular data support a sister group relationship between Troglocephalus rhinobatidis and Neoheterocotyle rhinobatidis to the exclusion of the other species of Neoheterocotyle and recognition of Troglocephalus renders Neoheterocotyle,le paraphyletic. We propose Troglocephalus incertae sedis. An updated classification and full species list of the Monocotylidae is provided. (C) 2001 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Recurrent gains and losses of large (84-109 bp) repeats in the rDNA internal transcribed spacer 2 (ITS2) of rhipicephaline ticks

We studied the internal transcribed spacer 2 (ITS2) in twenty-two spp. of ticks from the subfamily Rhipicephalinae. A 104-109 base pair (bp) region was Imperfectly repeated In most ticks studied. Mapping the number of repeat copies on to a phylogeny from the ITS2 showed that there have been many Independent gains and losses of repeats. Comparison of the sequences of the repeat copies Indicated that in most taxa concerted evolution had played little if any role in the evolution of these regions, as the copies clustered by sequence position rather than species, In our putative secondary structure, each repeat copy can fold into a distinct and almost identical stem-loop complex; a gain or loss of a repeat copy apparently does not impair the function of the ITS2 in these ticks.

A multiple-outgroup approach to resolving division-level phylogenetic relationships using 16S rDNA data

The 16S rRNA gene (16S rDNA) is currently the most widely used gene for estimating the evolutionary history of prokaryotes, To date, there are more than 30 000 16S rDNA sequences available from the core databases, GenBank, EMBL and DDBJ, This great number may cause a dilemma when composing datasets for phylogenetic analysis, since the choice and number of reference organisms are known to affect the resulting tree topology. A group of sequences appearing monophyletic in one dataset may not be so in another. This can be especially problematic when establishing the relationships of distantly related sequences at the division (phylum) level. In this study, a multiple-outgroup approach to resolving division-level phylogenetic relationships is suggested using 16S rDNA data. The approach is illustrated by two case studies concerning the monophyly of two recently proposed bacterial divisions, OP9 and OP10.

Foraging by the endangered black-breasted button-quail (Turnix melanogaster) within fragmented rainforest of an agricultural landscape

We studied the foraging habitat of the endangered black-breasted button-quail (Turnix melanogaster) in 13 rainforest patches of an agricultural landscape (23.4 km(2)) in eastern Australia to assess its use of fragmented habitats outside conservation reserves. The species foraged only in the three largest patches (17.4, 40.0, 63.8 ha in size), all of which were connected to open eucalypt forest, and in intact rainforest. Occurrence of birds was greatest in the largest patch. The maximum number of individuals within the study area was estimated to be 22. Radio-tracking of nine birds revealed that three were resident in the largest patch for periods of over 100 days; no movements between patches were detected. Three radio-tagged birds were taken by avian and mammalian predators. Our results indicated that the long-term future of the species in agricultural landscapes is bleak and that management action is urgently needed to arrest its decline in these ecosystems, (C) 2001 Elsevier Science Ltd. All rights reserved.