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1.  Molecular Phylogeny of Weakfish Species of the Stellifer Group (Sciaenidae, Perciformes) of the Western South Atlantic Based on Mitochondrial and Nuclear Data 
PLoS ONE  2014;9(7):e102250.
The phylogenetic relationships within the Stellifer group of weakfishes (Stellifer, Odontoscion, Ophioscion, and Bairdiella) were evaluated using 2723 base pairs comprising sequences of nuclear (rhodopsin, TMO-4C4, RAG-1) and mitochondrial (16S rRNA and COI) markers obtained from specimens of nine species. Our results indicate a close relationship between Bairdiella and Odontoscion, and also that the genus Stellifer is not monophyletic, but rather that it consists of two distinct lineages, one clade containing S. microps/S. naso/S. brasiliensis and the other, S. rastrifer/S. stellifer/Stellifer sp. B, which is closer to Ophioscion than the former clade. The O. punctatissimus populations from the northern and southern Brazilian coast were also highly divergent in both nuclear (0.8% for rhodopsin and 0.9% for RAG-1) and mitochondrial sequences (2.2% for 16S rRNA and 7.3% for COI), which we conclude is consistent with the presence of two distinct species. The morphological similarities of the members of the Stellifer group is reinforced by the molecular data from both the present study and previous analyses, which have questioned the taxonomic status of the Stellifer group. If, on the one hand, the group is in fact composed of four genera (Stellifer, Ophioscion, Odontoscion, and Bairdiella), one of the two Stellifer clades should be reclassified as a new genus. However, if the close relationship and the reduced genetic divergence found within the group is confirmed in a more extensive study, including representatives of additional taxa, this, together with the morphological evidence, would support downgrading the whole group to a single genus. Obviously, these contradictory findings reinforce the need for a more systematic taxonomic revision of the Stellifer group as a whole.
doi:10.1371/journal.pone.0102250
PMCID: PMC4094507  PMID: 25020128
2.  The Past and Present of an Estuarine-Resident Fish, the “Four-Eyed Fish” Anableps anableps (Cyprinodontiformes, Anablepidae), Revealed by mtDNA Sequences 
PLoS ONE  2014;9(7):e101727.
Historical events, such as changes in sea level during the Pleistocene glacial cycles, had a strong impact on coastal habitats, limiting connectivity and promoting the genetic divergence of various species. In this study, we evaluated the influence of climate oscillations and the possibility of estuary function as a barrier to gene flow among populations of the four-eyed fish, Anableps anableps. This species is fully estuarine-resident, has internal fertilization, is viviparous and does not migrate across long distances. These features make the four-eyed fish an excellent model for the study of evolutionary processes related to genetic differentiation of species and populations in estuaries. The evolutionary history of A. anableps was inferred from phylogeographic and population analyses using sequences of the mitochondrial DNA Control Region of 13 populations distributed in the Amazon and Northeast Coast of Brazil from Calcoene (Amapa) to Parnaiba (Piaui). The 83 retrieved haplotypes show a pattern of four distinct mitochondrial lineages, with up to 3.4% nucleotide divergence among them. The evolutionary reconstruction suggests that these lineages diverged recently in the late Pleistocene/early Holocene after the Atlantic Ocean reaching current levels. Analysis of variability, neutrality and the genetic expansion pattern revealed that the lineages have distinct characteristics, which were shaped by the different geomorphological features of coastal regions combined with sea level oscillations over a very long period of time. Only few neighboring populations show a discreet gene flow. This study may also be helpful for designing new experiments to better understand the geomorphological evolutionary history of the estuaries of the Amazon and the Northeast Coast of Brazil using estuarine-resident species as a model.
doi:10.1371/journal.pone.0101727
PMCID: PMC4086964  PMID: 25003185
3.  Molecular Phylogeny of the Genus Lolliguncula Steenstrup, 1881 Based on Nuclear and Mitochondrial DNA Sequences Indicates Genetic Isolation of Populations from North and South Atlantic, and the Possible Presence of Further Cryptic Species 
PLoS ONE  2014;9(2):e88693.
Squid of the genus Lolliguncula Steenstrup, 1881 are small bodied, coastal species capable of tolerating low salinity. Lolliguncula sp. are found exclusively in the New World, although only one of the four recognized species (Lolliguncula brevis) occurs in the Atlantic Ocean. Preliminary morphological analyses suggest that Lolliguncula brevis populations in the North and South Atlantic may represent distinct species. The principal objective of the present study was to verify the phylogenetic relationships within the genus and test for the presence of possible cryptic species. Both gene and species tree topologies indicated that Lolliguncula brevis specimens from the North and South Atlantic represent distinct phylogenetic clades. In contrast with previous studies, L. panamensis was identified as the basal species of the genus. Our results provide important insights into the phylogenetic relationships among the Lolliguncula specimens analyzed, and confirm the genetic separation of Lolliguncula brevis populations of the North and South Atlantic at the level of sister species.
doi:10.1371/journal.pone.0088693
PMCID: PMC3934857  PMID: 24586371
5.  New molecular evidence supports the species status of Kaempfer’s Woodpecker (Aves, Picidae) 
Genetics and Molecular Biology  2013;36(2):192-200.
Kaempfer’s Woodpecker (Celeus obrieni) is the only species of the genus Celeus endemic to Brazil. The description of this taxon as a subspecies of the Rufous-headed Woodpecker (Celeus spectabilis) was based on a single specimen. While C. obrieni and C. spectabilis are now considered separate species based on morphological and limited molecular evidence, no study has critically tested the reciprocal monophyly and degree of evolutionary independence between these taxa with several specimens. Herein, fragments of the mitochondrial and nuclear DNA of three recently-collected specimens of C. obrieni were analyzed to evaluate the degree of evolutionary differentiation of this taxon with respect to C. spectabilis. The results confirm the reciprocal monophyly between the specimens of C. obrieni and C. spectabilis. The genetic divergence values for the two taxa also support their classification as independent species, given that they are greater than the values recorded among other closely-related but separate species of the same genus. Estimates of the divergence time between C. obrieni and C. spectabilis indicate that cladogenesis occurred in the mid-Pleistocene, during a period of major climatic fluctuations and landscape change, consistent with the hypothesis of a corridor of open bamboo dominated forests and woodland stretching.
doi:10.1590/S1415-47572013005000022
PMCID: PMC3715285  PMID: 23885201
Bamboo; biogeography; Celeus obrieni; Celeus spectabilis; taxonomy
6.  The Detection and Sequencing of a Broad-Host-Range Conjugative IncP-1β Plasmid in an Epidemic Strain of Mycobacterium abscessus subsp. bolletii 
PLoS ONE  2013;8(4):e60746.
Background
An extended outbreak of mycobacterial surgical infections occurred in Brazil during 2004–2008. Most infections were caused by a single strain of Mycobacterium abscessus subsp. bolletii, which was characterized by a specific rpoB sequevar and two highly similar pulsed-field gel electrophoresis (PFGE) patterns differentiated by the presence of a ∼50 kb band. The nature of this band was investigated.
Methodology/Principal Findings
Genomic sequencing of the prototype outbreak isolate INCQS 00594 using the SOLiD platform demonstrated the presence of a 56,264-bp circular plasmid, designated pMAB01. Identity matrices, genetic distances and phylogeny analyses indicated that pMAB01 belongs to the broad-host-range plasmid subgroup IncP-1β and is highly related to BRA100, pJP4, pAKD33 and pB10. The presence of pMAB01-derived sequences in 41 M. abscessus subsp. bolletii isolates was evaluated using PCR, PFGE and Southern blot hybridization. Sixteen of the 41 isolates showed the presence of the plasmid. The plasmid was visualized as a ∼50-kb band using PFGE and Southern blot hybridization in 12 isolates. The remaining 25 isolates did not exhibit any evidence of this plasmid. The plasmid was successfully transferred to Escherichia coli by conjugation and transformation. Lateral transfer of pMAB01 to the high efficient plasmid transformation strain Mycobacterium smegmatis mc2155 could not be demonstrated.
Conclusions/Significance
The occurrence of a broad-host-range IncP-1β plasmid in mycobacteria is reported for the first time. Thus, genetic exchange could result in the emergence of specific strains that might be better adapted to cause human disease.
doi:10.1371/journal.pone.0060746
PMCID: PMC3614916  PMID: 23565273
7.  Dispersal Capacity and Genetic Structure of Arapaima gigas on Different Geographic Scales Using Microsatellite Markers 
PLoS ONE  2013;8(1):e54470.
Despite the ecological and economic importance of the Arapaima gigas (Cuvier 1817), few data about its dispersal capacity are available. The present study was based on the analysis of microsatellite markers in order to estimate the dispersal capacity of the species on fine, meso, and large geographic scales. For this, 561 specimens obtained from stocks separated by distances of up to 25 km (fine scale), 100 km (meso scale), and 1300–2300 km (large scale) were analyzed. The fine scale analysis indicated a marked genetic similarity between lakes, with low genetic differentiation, and significant differences between only a few pairs of sites. Low to moderate genetic differentiation was observed between pairs of sites on a meso scale (100 km), which could be explained by the distances between sites. By contrast, major genetic differentiation was recorded in the large scale analysis, that is, between stocks separated by distances of over 1300 km, with the analysis indicating that differentiation was not related solely to distance. The genetic structuring analysis indicated the presence of two stocks, one represented by the arapaimas of the Mamirauá Reserve, and the other by those of Santarém and Tucuruí. The dispersal of arapaimas over short distances indicates a process of lateral migration within the várzea floodplains, which may be the principal factor determining the considerable homogeneity observed among the várzea lakes. The populations separated by distances of approximately 100 km were characterized by reduced genetic differentiation, which was associated with the geographic distances between sites. Populations separated by distances of over 1300 km were characterized by a high degree of genetic differentiation, which may be related primarily to historical bottlenecks in population size and the sedentary behavior of the species. Evidence was found of asymmetric gene flow, resulting in increasing genetic variability in the population of the Mamirauá Reserve.
doi:10.1371/journal.pone.0054470
PMCID: PMC3553164  PMID: 23372730
8.  Inclusion of South American samples reveals new population structuring of the blacktip shark (Carcharhinus limbatus) in the western Atlantic 
Genetics and Molecular Biology  2012;35(4):752-760.
Carcharhinus limbatus has a cosmopolitan distribution and marked genetic structuring, mainly because of its philopatric behavior. However, analysis of this structuring has not previously included South American populations. In the present study, we analyzed a sample of adult individuals collected on the northern coast of Brazil and compared the sequences of the mitochondrial control region with those of populations already genotyped. Relatively high haplotype diversity (12 haplotypes, genetic diversity of 0.796) was observed, similar to that in other populations but with a much larger number of private alleles. In contrast to populations studied previously, which were represented by neonates, the pronounced allelic variability found in the South American individuals may have resulted from migrations from other populations in the region that have yet to be genotyped. This population was also genetically distinct from the other Atlantic populations (Fst > 0.8), probably because of female philopatry, and apparently separated from the northwestern Atlantic group 1.39 million years ago. These findings indicate that the C. limbatus population from northern Brazil is genetically distinct from all other populations and should be considered as a different management unit for the protection of stocks.
doi:10.1590/S1415-47572012005000062
PMCID: PMC3526082  PMID: 23271935
Carcharhinus limbatus; control region; fishery stocks; mitochondrial DNA; population genetics
9.  High-throughput sequencing of black pepper root transcriptome 
BMC Plant Biology  2012;12:168.
Background
Black pepper (Piper nigrum L.) is one of the most popular spices in the world. It is used in cooking and the preservation of food and even has medicinal properties. Losses in production from disease are a major limitation in the culture of this crop. The major diseases are root rot and foot rot, which are results of root infection by Fusarium solani and Phytophtora capsici, respectively. Understanding the molecular interaction between the pathogens and the host’s root region is important for obtaining resistant cultivars by biotechnological breeding. Genetic and molecular data for this species, though, are limited. In this paper, RNA-Seq technology has been employed, for the first time, to describe the root transcriptome of black pepper.
Results
The root transcriptome of black pepper was sequenced by the NGS SOLiD platform and assembled using the multiple-k method. Blast2Go and orthoMCL methods were used to annotate 10338 unigenes. The 4472 predicted proteins showed about 52% homology with the Arabidopsis proteome. Two root proteomes identified 615 proteins, which seem to define the plant’s root pattern. Simple-sequence repeats were identified that may be useful in studies of genetic diversity and may have applications in biotechnology and ecology.
Conclusions
This dataset of 10338 unigenes is crucially important for the biotechnological breeding of black pepper and the ecogenomics of the Magnoliids, a major group of basal angiosperms.
doi:10.1186/1471-2229-12-168
PMCID: PMC3487918  PMID: 22984782
10.  Hybridization and massive mtDNA unidirectional introgression between the closely related Neotropical toads Rhinella marina and R. schneideri inferred from mtDNA and nuclear markers 
Background
The classical perspective that interspecific hybridization in animals is rare has been changing due to a growing list of empirical examples showing the occurrence of gene flow between closely related species. Using sequence data from cyt b mitochondrial gene and three intron nuclear genes (RPL9, c-myc, and RPL3) we investigated patterns of nucleotide polymorphism and divergence between two closely related toad species R. marina and R. schneideri. By comparing levels of differentiation at nuclear and mtDNA levels we were able to describe patterns of introgression and infer the history of hybridization between these species.
Results
All nuclear loci are essentially concordant in revealing two well differentiated groups of haplotypes, corresponding to the morphologically-defined species R. marina and R. schneideri. Mitochondrial DNA analysis also revealed two well-differentiated groups of haplotypes but, in stark contrast with the nuclear genealogies, all R. schneideri sequences are clustered with sequences of R. marina from the right Amazon bank (RAB), while R. marina sequences from the left Amazon bank (LAB) are monophyletic. An Isolation-with-Migration (IM) analysis using nuclear data showed that R. marina and R. schneideri diverged at ≈ 1.69 Myr (early Pleistocene), while R. marina populations from LAB and RAB diverged at ≈ 0.33 Myr (middle Pleistocene). This time of divergence is not consistent with the split between LAB and RAB populations obtained with mtDNA data (≈ 1.59 Myr), which is notably similar to the estimate obtained with nuclear genes between R. marina and R. schneideri. Coalescent simulations of mtDNA phylogeny under the speciation history inferred from nuclear genes rejected the hypothesis of incomplete lineage sorting to explain the conflicting signal between mtDNA and nuclear-based phylogenies.
Conclusions
The cytonuclear discordance seems to reflect the occurrence of interspecific hybridization between these two closely related toad species. Overall, our results suggest a phenomenon of extensive mtDNA unidirectional introgression from the previously occurring R. schneideri into the invading R. marina. We hypothesize that climatic-induced range shifts during the Pleistocene/Holocene may have played an important role in the observed patterns of introgression.
doi:10.1186/1471-2148-11-264
PMCID: PMC3192708  PMID: 21939538
11.  The history of the introduction of the giant river prawn, Macrobrachium cf. rosenbergii (Decapoda, Palaemonidae), in Brazil: New insights from molecular data 
Genetics and Molecular Biology  2011;34(1):142-151.
The giant river prawn, Macrobrachium cf. rosenbergii, is one of the most cultivated freshwater prawns in the world and has been introduced into more than 40 countries. In some countries, this prawn is considered an invasive species that requires close monitoring. Recent changes in the taxonomy of this species (separation of M. rosenbergii and M. dacqueti) require a re-evaluation of introduced taxa. In this work, molecular analyses were used to determine which of these two species was introduced into Brazil and to establish the geographic origin of the introduced populations that have invaded Amazonian coastal waters. The species introduced into Brazil was M. dacqueti through two introduction events involving prawns originating from Vietnam and either Bangladesh or Thailand. These origins differ from historical reports of the introductions and underline the need to confirm the origin of other exotic populations around the world. The invading populations in Amazonia require monitoring not only because the biodiversity of this region may be affected by the introduction, but also because admixture of different native haplotypes can increase the genetic variability and the likelihood of persistence of the invading species in new habitats.
doi:10.1590/S1415-47572010005000115
PMCID: PMC3085361  PMID: 21637558
Bioinvasion; exotic species; giant river prawn; Macrobrachium dacqueti; COI; 16S rRNA; Amazonia
12.  Molecular identification, phylogeny and geographic distribution of Brazilian mangrove oysters (Crassostrea) 
Genetics and Molecular Biology  2010;33(3):564-572.
Oysters (Ostreidae) manifest a high degree of phenotypic plasticity, whereby morphology is of limited value for species identification and taxonomy. By using molecular data, the aim was to genetically characterize the species of Crassostrea occurring along the Brazilian coast, and phylogenetically relate these to other Crassostrea from different parts of the world. Sequencing of the partial cytochrome oxidase c subunit I gene (COI), revealed a total of three species of Crassostrea at 16 locations along the Brazilian coast. C. gasar was found from Curuçá (Pará state) to Santos (São Paulo state), and C. rhizophorae from Fortim (Ceará state) to Florianópolis (Santa Catarina state), although small individuals of the latter species were also found at Ajuruteua beach (municipality of Bragança, Pará state). An unidentified Crassostrea species was found only on Canela Island, Bragança. Crassostrea gasar and C. rhizophorae grouped with C. virginica, thereby forming a monophyletic Atlantic group, whereas Crassostrea sp. from Canela Island was shown to be more similar to Indo-Pacific oysters, and either arrived in the Atlantic Ocean before the convergence of the Isthmus of Panama or was accidentally brought to Brazil by ship.
doi:10.1590/S1415-47572010000300030
PMCID: PMC3036104  PMID: 21637433
genetic identification; oysters; biogeography; COI gene; Ostreidae
13.  Genetic characterization of native and introduced populations of the neotropical cichlid genus Cichla in Brazil 
Genetics and Molecular Biology  2009;32(3):601-607.
A molecular phylogenetic analysis based on mitochondrial 16S ribosomal DNA and Control Region sequences from native and introduced populations was undertaken, in order to characterize the introduction of Cichla (peacock bass or tucunaré) species in Brazil. Mitochondrial DNA haplotypes found in introduced fish from Minas Gerais state (southeastern Brazil) clustered only with those from native species of the Tocantins River (Cichla piquiti and C. kelberi), thereby suggesting a single or, at most, few translocation acts in this area, even though with fish from the same source-population. Our study contributes to an understanding of the introduction of Cichla in regions of Brazil outside the Amazon basin, and adds phylogenetic data to the recently describe Cichla species, endemic from the Tocantins-Araguaia basin.
doi:10.1590/S1415-47572009005000060
PMCID: PMC3036039  PMID: 21637525
peacock bass or tucunaré; mtDNA; invasive populations; Amazon basin; Minas Gerais
14.  Identification and phylogenetic inferences on stocks of sharks affected by the fishing industry off the Northern coast of Brazil 
Genetics and Molecular Biology  2009;32(2):405-413.
The ongoing decline in abundance and diversity of shark stocks, primarily due to uncontrolled fishery exploitation, is a worldwide problem. An additional problem for the development of conservation and management programmes is the identification of species diversity within a given area, given the morphological similarities among shark species, and the typical disembarkation of processed carcasses which are almost impossible to differentiate. The main aim of the present study was to identify those shark species being exploited off northern Brazil, by using the 12S-16S molecular marker. For this, DNA sequences were obtained from 122 specimens collected on the docks and the fish market in Bragança, in the Brazilian state of Pará. We identified at least 11 species. Three-quarters of the specimens collected were either Carcharhinus porosus or Rhizoprionodon sp, while a notable absence was the daggernose shark, Isogomphodon oxyrhyncus, previously one of the most common species in local catches. The study emphasises the value of molecular techniques for the identification of cryptic shark species, and the potential of the 12S-16S marker as a tool for phylogenetic inferences in a study of elasmobranchs.
doi:10.1590/S1415-47572009005000039
PMCID: PMC3036939  PMID: 21637699
sharks; molecular identification; phylogenetic; mtDNA; conservation

Results 1-14 (14)