Unusually short tandem repeats appear to reach chromosome ends of Rhynchosciara americana (Diptera: Sciaridae)

The characterisation of sequences at chromosome ends of Rhynchosciara americana was continued with the screening of a genomic library using as a probe a short repeat identified in a previous report (M-22, 22 bp) which was found to be specific for noncentromeric termini of this species. Simple repeats, complex tandem and apparently dispersed repeats were present in the genomic clones analysed. Repetitive sequences do not define individual chromosome tips as they were found in all noncentromeric ends. A novel and unusually short tandem repeat type for dipteran chromosome ends (named M-16) composed of 16 nucleotides and frequently associated with M-22 arrays was characterised in this work. Islands of M-16 and M-22 tandem repeats were found in all the genomic clones analysed. Individual probes representative of each repetitive element hybridised not only to all noncentromeric ends of R. americana chromosomes but also to inter-telomeric bridges. This contrasted with the other repeat types which displayed sub-telomeric localisation as seen by double detection of hybridised probe and telomeric reverse transcriptase. Some stretches composed of M-16 and M-22 tandem repeats localised in different regions of the analysed genomic clones were either identical or showed sequence similarity that was unexpectedly higher than the mean sequence similarity observed among repeats within each of their tandem arrays. The occurrence of segmental duplications, as deduced by sequence analyses involving the two repeats that appeared to reach chromosome ends, might indicate the involvement of this type of duplication process in the chromosome end maintenance in this species.

Role of the Mitochondrial Mutations, m. 827A > G and the Novel m. 7462C > T, in the Origin of Hearing Loss

Samples from 30 deaf probands exhibiting features suggestive of syndromic mitochondrial deafness or from families with maternal transmission of deafness were selected for investigation of mutations in the mitochondrial genes MT-RNR1 and MT-TS1. Patients with mutation m. 1555A>G had been previously excluded from this sample. In the MT-RNR1 gene, five probands presented the m. 827A>G sequence variant, of uncertain pathogenicity. This change was also detected in 66 subjects of an unaffected control sample of 306 Brazilian individuals from various ethnic backgrounds. Given its high frequency, we consider it unlikely to have a pathogenic role on hereditary deafness. As to the MT-TS1 gene, one proband presented the previously known pathogenic m. 7472insC mutation and three probands presented a novel variant, m. 7462C>T, which was absent from the same control sample of 306 individuals. Because of its absence in control samples and association with a family history of hearing impairment, we suggest it might be a novel pathogenic mutation.

Genetic structure, phylogeny, and biogeography of Brazilian eyelid-less lizards of genera Calyptommatus and Nothobachia (Squamata, Gymnophthalmidae) as inferred from mitochondrial DNA sequences

Calyptommatus and Nothobachia genera of gymnophthalmid lizards are restricted to sandy open habitats on Sao Francisco River margins, northeastern Brazil. Phylogenetic relationships and geographic distribution of the four recognized species of Calyptommatus were analyzed from partial mitochondrial cyt b, 12S, and 16S rRNA genes sequencing, taking allopatric populations of the monotypic Nothobachia ablephara as the outgroup. In Calyptommatus a basal split separated C. sinebrachiatus, a species restricted to the eastern bank of the river, from the three other species. In this clade, C. confusionibus, found on western margin, was recovered as the sister group of the two other species, C. leiolepis and C. nicterus, from opposite margins. According to approximate date estimations, C. sinebrachiatus would have separated from the other congeneric species by 4.4-6.5 my, and C. nicterus, also from eastern bank, would be diverging by 1.8-2.6 my from C. leiolepis, the sister species on the opposite margin. C. confusionibus and C. leiolepis, both from western sandy areas, would be differentiating by 2.8-5.0 my. Divergence times of about 3.0-4.0 my were estimated for allopatric populations of Nothobachia restricted to western margin. Significant differences in 16S rRNA secondary structure relatively to other vertebrates are reported. Distinct evolutionary patterns are proposed for different taxa in those sandy areas, probably related to historical changes in the course of Sao Francisco River. (C) 2010 Elsevier Inc. All rights reserved.

Curiously composite structures of a retrotransposon and a complex repeat associated with chromosome ends of Rhynchosciara americana (Diptera: Sciaridae)

In Drosophila, telomere retrotransposons counterbalance the loss of telomeric DNA. The exceptional mechanism of telomere recovery characterized in Drosophila has not been found in lower dipterans (Nematocera). However, a retroelement resembling a telomere transposon and termed ""RaTART"" has been described in the nematoceran Rhynchosciara americana. In this work, DNA and protein sequence analyses, DNA cloning, and chromosomal localization of probes obtained either by PCR or by screening a genomic library were carried out in order to examine additional features of this retroelement. The analyses performed raise the possibility that RaTART represents a genomic clone composed of distinct repetitive elements, one of which is likely to be responsible for its apparent enrichment at chromosome ends. RaTART sequence in addition allowed to assess a novel subtelomeric region of R. americana chromosomes that was analyzed in this work after subcloning a DNA fragment from a phage insert. It contains a complex repeat that is located in the vicinity of simple and complex tandem repeats characterized previously. Quantification data suggest that the copy number of the repeat is significantly lower than that observed for the ribosomal DNA in the salivary gland of R. americana. A short insertion of the RaTART was identified in the cloned segment, which hybridized preferentially to subtelomeres. Like RaTART, it displays truncated sequences related to distinct retrotransposons, one of which has a conceptual translation product with significant identity with an endonuclease from a lepidopteran retrotransposon. The composite structure of this DNA stretch probably reflects mobile element activity in the subtelomeric region analyzed in this work.

Comparative Genetic Diversity of Wild and Captive Populations of the Bare-Faced Curassow (Crax fasciolata) Based on Cross-Species Microsatellite Markers: Implications for Conservation and Management

The bare-faced curassow (Crax fasciolata) is a large Neotropical bird that suffers anthropogenic pressure across much of its range. A captive population is maintained for conservation management, although there has been no genetic screening of stocks. Based on the six microsatellite markers developed for Crax globulosa, the genetic variability of C. fasciolata and possible differences between a wild and a captive population were investigated. Only three loci were polymorphic, with a total of 27 alleles. More than half of these alleles were private to the wild (n = 8) or captive (n = 7) populations. Significant deviations from Hardy-Weinberg equilibrium were restricted to the captive population. Despite the number of private alleles, genetic drift has probably promoted differentiation between populations. Our results indicate that wild C. fasciolata populations are genetically impoverished and structured, but species-specific microsatellite markers will be necessary for a more reliable assessment of the species` genetic diversity.

Mitochondrial DNA Diversity of Orchid Bee Euglossa fimbriata (Hymenoptera: Apidae) Populations Assessed by PCR-RFLP

Euglossa fimbriata is a euglossine species widely distributed in Brazil and occurring primarily in Atlantic Forest remnants. In this study, the genetic mitochondrial structure of E. fimbriata from six Atlantic Forest fragments was studied by RFLP analysis of three PCR-amplified mtDNA gene segments (16S, COI-COII, and cyt b). Ten composite haplotypes were identified, six of which were exclusive and represented singleton mitotypes. Low haplotype diversity (0.085-0.289) and nucleotide diversity (0.000-0.002) were detected within samples. AMOVA partitioned 91.13% of the overall genetic variation within samples and 8.87% (I center dot(st) = 0.089; P < 0.05) among samples. Pairwise comparisons indicated high levels of differentiation among some pairs of samples (I center dot(st) = 0.161-0.218; P < 0.05). These high levels indicate that these populations of E. fimbriata, despite their highly fragmented landscape, apparently have not suffered loss of genetic variation, suggesting that this particular population is not currently endangered.

The timing of Neotropical speciation dynamics: A reconstruction of Myiopagis flycatcher diversification using phylogenetic and paleogeographic data

Neotropical forests have brought forth a large proportion of the world`s terrestrial biodiversity, but the underlying evolutionary mechanisms and their timing require further elucidation. Despite insights gained from phylogenetic studies, uncertainties about molecular clock rates have hindered efforts to determine the timing of diversification processes. Moreover, most molecular research has been detached from the extensive body of data on Neotropical geology and paleogeography. We here examine phylogenetic relationships and the timing of speciation events in a Neotropical flycatcher genus (Myiopagis) by using calibrations from modern geologic data in conjunction with a number of recently developed DNA sequence dating algorithms and by comparing these estimates with those based on a range of previously proposed molecular clock rates. We present a well-supported hypothesis of systematic relationships within the genus. Our age estimates of Myiopagis speciation events based on paleogeographic data are in close agreement with nodal ages derived from a ""traditional"" avian mitochondrial 2%/My clock, while contradicting other clock rates. Our comparative approach corroborates the consistency of the traditional avian mitochondrial clock rate of 2%/My for tyrant-flycatchers. Nevertheless, our results argue against the indiscriminate use of molecular clock rates in evolutionary research and advocate the verification of the appropriateness of the traditional clock rate by means of independent calibrations in individual studies. (C) 2009 Elsevier Inc. All rights reserved.

Chromosome homologies of the highly rearranged karyotypes of four Akodon species (Rodentia, Cricetidae) resolved by reciprocal chromosome painting: the evolution of the lowest diploid number in rodents

Traditionally comparative cytogenetic studies are based mainly on banding patterns. Nevertheless, when dealing with species with highly rearranged genomes, as in Akodon species, or with other highly divergent species, cytogenetic comparisons of banding patterns prove inadequate. Hence, comparative chromosome painting has become the method of choice for genome comparisons at the cytogenetic level since it allows complete chromosome probes of a species to be hybridized in situ onto chromosomes of other species, detecting homologous genomic regions between them. In the present study, we have explored the highly rearranged complements of the Akodon species using reciprocal chromosome painting through species-specific chromosome probes obtained by chromosome sorting. The results revealed complete homology among the complements of Akodon sp. n. (ASP), 2n = 10; Akodon cursor (ACU), 2n = 15; Akodon montensis (AMO), 2n = 24; and Akodon paranaensis (APA), 2n = 44, and extensive chromosome rearrangements have been detected within the species with high precision. Robertsonian and tandem rearrangements, pericentric inversions and/or centromere repositioning, paracentric inversion, translocations, insertions, and breakpoints, where chromosomal rearrangements, seen to be favorable, were observed. Chromosome painting using the APA set of 21 autosomes plus X and Y revealed eight syntenic segments that are shared with A. montensis, A. cursor, and ASP, and one syntenic segment shared by A. montensis and A. cursor plus five exclusive chromosome associations for A. cursor and six for ASP chromosome X, except for the heterochromatin region of ASP X, and even chromosome Y shared complete homology among the species. These data indicate that all those closely related species have experienced a recent extensive process of autosomal rearrangement in which, except for ASP, there is still complete conservation of sex chromosomes homologies.

Phylogeny of the Neotropical genus Acestrorhynchus (Ostariophysi: Characiformes) based on nuclear and mitochondrial gene sequences and morphology: A total evidence approach

Acestrorhynchus is the sole genus of the family Acestrorhynchidae which includes 14 species currently recognized as valid. Species of Acestrorhynchus comprise small-to-medium sized piscivorous fishes and have been traditionally grouped on the basis of well-defined color patterns. A recent phylogeny, based on morphological characters, could not resolve the phylogenetic affinities of A. heterolepis and the relationships among the species of the clade formed by A. abbreviatus, A. altus, A. falcatus, A. lacustris, and A. pantaneiro. The simultaneous analysis of two mitochondrial genes (16S and ATP synthase subunits 6 and 8) and one nuclear intron (S7) was able to resolve the latter clade, but the position of A. heterolepis remained unresolved. The combination of the molecular and morphological data sets in a total evidence analysis resulted in a well-resolved hypothesis regarding the phylogenetic relationships of Acestrorhynchus species. (C) 2009 Elsevier Inc. All rights reserved.

Potential sites of triple-helical nucleic acid formation in chromosomes of Rhynchosciara (Diptera: Sciaridae) and Drosophila melanogaster

Antibodies to specific nucleic acid conformations are amongst the methods that have allowed the study of non-canonical (Watson-Crick) DNA structures in higher organisms. In this work, the structural limitations for the immunological detection of DNA.RNA hybrid duplexes were examined using specific RNA homopolymers as probes for homopolymer polydeoxyadenylic acid (poly(dA)).polydeoxythymidylic acid (poly(dT))-rich regions of Rhynchosciara americana (Diptera: Sciaridae) chromosomes. Anti-DNA.RNA duplexes did not react with the complex formed between chromosomal poly(dA) and exogenous polyuridylic acid (poly(rU)). Additionally, poly(rU) prevented the detection of polyadenylic acid.poly(dT) hybrid duplexes preformed in situ. These results raised the possibility that three-stranded structures rather than duplexes were formed in chromosomal sites. To test this hypothesis, the specificity of antibodies to triple-helical nucleic acids was reassessed employing distinct nucleic acid configurations. These antibodies were raised to the poly(dA).poly(rU).poly(rU) complex and have been used here for the first time in immunocytochemistry. Anti-triplex antibodies recognised the complex poly(dA).poly(rU).poly(rU) assembled with poly(rU) in poly(dA).poly(dT)-rich homopolymer regions of R. americana chromosomes. The antibodies could not detect short triplex stretches, suggesting the existence of constraints for triple-helix detection, probably related to triplex tract length. In addition, anti-poly(dA).poly(rU).poly(rU) antibodies reacted with the pericentric heterochromatin of RNase-treated polytene chromosomes of R. americana and Drosophila melanogaster. In apparent agreement with data obtained in cell types from other organisms, the results of this work suggest that significant triple-helix DNA extensions can be formed in pericentric regions of these species.

Ribosomal RNA gene insertions in the R2 site of Rhynchosciara (Diptera: Sciaridae)

Ribosomal RNA genes of most insects are interrupted by R1/R2 retrotransposons. The occurrence of R2 retrotransposons in sciarid genomes was studied by PCR and Southern blot hybridization in three Rhynchosciara species and in Trichosia pubescens. Amplification products with the expected size for non-truncated R2 elements were only obtained in Rhynchosciara americana. The rDNA in this species is located in the proximal end of the X mitotic chromosome but in the salivary gland is associated with all four polytene chromosomes. Approximately 50% of the salivary gland rDNA of most R. americana larval groups analysed had an insertion in the R2 site, while no evidence for the presence of R1 elements was found. In-situ hybridization results showed that rDNA repeat units containing R2 take part in the structure of the extrachromosomal rDNA. Also, rDNA resistance to Bal 31 digestion could be interpreted as evidence for nonlinear rDNA as part of the rDNA in the salivary gland. Insertions in the rDNA of three other sciarid species were not detected by Southern blot and in-situ hybridization, suggesting that rDNA retrotransposons are significantly under-represented in their genomes in comparison with R. americana. R2 elements apparently restricted to R. americana correlate with an increased amount of repetitive DNA in its genome in contrast to other Rhynchosciara species. The results obtained in this work together with previous results suggest that evolutionary changes in the genus Rhynchosciara occurred by differential genomic occupation not only of satellite DNA but possibly also of rDNA retrotransposons.

The hidden diversity of Coleodactylus amazonicus (Sphaerodactylinae, Gekkota) revealed by molecular data

Coleodactylus amazonicus, a small leaf-litter diurnal gecko widely distributed in Amazon Basin has been, considered a single species with no significant morphological differences between populations along its range. A recent molecular study, however, detected large genetic differences between populations of central Amazonia and those in the easternmost part of the Amazon Basin, suggesting the presence of taxonomically unrecognised diversity. In this study, DNA sequences of three mitochondrial (165, cytb, and ND4) and two nuclear genes (RAG-1, c-mos) were used to investigate whether the species currently identified as C. amazonicus contains morphologically cryptic species lineages. The present phylogenetic analysis reveals further genetic subdivision including at least five potential species lineages, restricted to northeastern (lineage A), southeastern (lineage B), central-northern (lineage E) and central-southern (lineages C and D) parts of Amazon Basin. All clades are characterized by exclusive groups of alleles for both nuclear genes and highly divergent mitochondrial haplotype clades, with corrected pairwise net sequence divergence between sister lineages ranging from 9.1% to 20.7% for the entire mtDNA dataset. Results of this study suggest that the real diversity of ""C. amazonicus"" has been underestimated due to its apparent cryptic diversification. (C) 2009 Elsevier Inc. All rights reserved.

Complex meiotic configuration of the holocentric chromosomes: the intriguing case of the scorpion Tityus bahiensis

Mitotic and meiotic chromosomes of Tityus bahiensis were investigated using light (LM) and transmission electron microscopy (TEM) to determine the chromosomal characteristics and disclose the mechanisms responsible for intraspecific variability in chromosome number and for the presence of complex chromosome association during meiosis. This species is endemic to Brazilian fauna and belongs to the family Buthidae, which is considered phylogenetically basal within the order Scorpiones. In the sample examined, four sympatric and distinct diploid numbers were observed: 2n = 5, 2n = 6, 2n = 9, and 2 = 10. The origin of this remarkable chromosome variability was attributed to chromosome fissions and/or fusions, considering that the decrease in chromosome number was concomitant with the increase in chromosome size and vice versa. The LM and TEM analyses showed the presence of chromosomes without localised centromere, the lack of chiasmata and recombination nodules in male meiosis, and two nucleolar organiser regions carrier chromosomes. Furthermore, male prophase I cells revealed multivalent chromosome associations and/or unsynapsed or distinctly associated chromosome regions (gaps, less-condensed chromatin, or loop-like structure) that were continuous with synapsed chromosome segments. All these data permitted us to suggest that the chromosomal rearrangements of T. bahiensis occurred in a heterozygous state. A combination of various factors, such as correct disjunction and balanced segregation of the chromosomes involved in complex meiotic pairing, system of achiasmate meiosis, holocentric nature of the chromosomes, population structure, and species dispersion patterns, could have contributed to the high level of chromosome rearrangements present in T. bahiensis.

The genus Coleodactylus (Sphaerodactylinae, Gekkota) revisited: A molecular phylogenetic perspective

Nucleotide sequence data from a mitochondrial gene (16S) and two nuclear genes (c-mos, RAG-1) were used to evaluate the monophyly of the genus Coleodactylus, to provide the first phylogenetic hypothesis of relationships among its species in a cladistic framework, and to estimate the relative timing, of species divergences. Maximum Parsimony, Maximum Likelihood and Bayesian analyses of the combined data sets retrieved Coleodactylus as a monophyletic genus, although weakly Supported. Species were recovered as two genetically and morphological distinct clades, with C. amazonicus populations forming the sister taxon to the meridionalis group (C. brachystoma, C. meridionalis, C. natalensis, and C. septentrionalis). Within this group, C. septentrionalis was placed as the sister taxon to a clade comprising the rest of the species, C. meridionalis was recovered as the sister species to C. brachystoma, and C natalensis was found nested within C. meridionalis. Divergence time estimates based on penalized likelihood and Bayesian dating methods do not Support the previous hypothesis based on the Quaternary rain forest fragmentation model proposed to explain the diversification of the genus. The basal cladogenic event between major lineages of Coleodactylus was estimated to have occurred in the late Cretaceous (72.6 +/- 1.77 Mya), approximately at the same point in time than the other genera of Sphaerodactylinae diverged from each other. Within the meridionalis group, the split between C. septentrionalis and C. brachystoma + C. meridionalis was placed in the Eocene (46.4 +/- 4.22 Mya), and the divergence between C. brachystoma and C. meridionalis was estimated to have occurred in the Oligocene (29.3 +/- 4.33 Mya). Most intraspecific cladogenesis occurred through Miocene to Pliocene, and only for two conspecific samples and for C. natalensis could a Quaternary differentiation be assumed (1.9 +/- 1.3 Mya). (C) 2008 Elsevier Inc. All rights reserved.

Mitochondrial sequence data and Dipsacales phylogeny: Mixed models, partitioned Bayesian analyses, and model selection

Phylogenetic analyses of chloroplast DNA sequences, morphology, and combined data have provided consistent support for many of the major branches within the angiosperm, clade Dipsacales. Here we use sequences from three mitochondrial loci to test the existing broad scale phylogeny and in an attempt to resolve several relationships that have remained uncertain. Parsimony, maximum likelihood, and Bayesian analyses of a combined mitochondrial data set recover trees broadly consistent with previous studies, although resolution and support are lower than in the largest chloroplast analyses. Combining chloroplast and mitochondrial data results in a generally well-resolved and very strongly supported topology but the previously recognized problem areas remain. To investigate why these relationships have been difficult to resolve we conducted a series of experiments using different data partitions and heterogeneous substitution models. Usually more complex modeling schemes are favored regardless of the partitions recognized but model choice had little effect on topology or support values. In contrast there are consistent but weakly supported differences in the topologies recovered from coding and non-coding matrices. These conflicts directly correspond to relationships that were poorly resolved in analyses of the full combined chloroplast-mitochondrial data set. We suggest incongruent signal has contributed to our inability to confidently resolve these problem areas. (c) 2007 Elsevier Inc. All rights reserved.