Hydrography rather than lip morphology better explains the evolutionary relationship between Gymnogeophagus labiatus and G. lacustris in Southern Brazil (Cichlidae: Geophagini)

Gymnogeophagus labiatus and G. lacustris have been long recognized as sister species exhibiting different ecological requirements. Gymnogeophagus labiatus occurs in rock bottom rivers in the hydrographic basins of Patos Lagoon (HBP) and Tramandaí River (HBT), while G. lacustris is exclusive from sand bottom coastal lagoons of the HBT. In this study, we used molecular markers, morphological measurements and data from nuptial male coloration to investigate the evolutionary relationship between these species in each hydrographic basin. We found, for all data sets, a closer relationship between G. labiatus and G. lacustris from the HBT than between G. labiatus populations from HBT and HBP. In particular, lip area had a large intraspecific plasticity, being uninformative to diagnose G. lacustris from G. labiatus. Molecular clock-based estimates suggest a recent divergence between species in the HBT (17,000 years ago), but not between G. labiatus from HBP and HBT (3.6 millions of years ago). Finally, we also found a divergent G. labiatus genetic lineage from the Camaquã River, in the HBP. These results show that the current taxonomy of G. labiatus and G. lacustris does not properly represent evolutionary lineages in these species.(AU)

Gymnogeophagus labiatus e G. lacustris vêm sendo consideradas espécies irmãs que possuem diferentes exigências ecológicas. Gymnogeophagus labiatus ocorre em rios de fundo de pedra nas bacias hidrográficas da Laguna dos Patos (HBP) e do rio Tramandaí (HBT), enquanto G. lacustris é exclusivo da HBT, ocorrendo em lagoas costeiras de fundo de arenoso. Nesse estudo, foram usados marcadores moleculares, medidas morfológicas e dados sobre a coloração nupcial em machos para investigar a relação evolutiva entre estas espécies em cada bacia hidrográfica. Para todos os conjuntos de dados foi observada uma relação mais próxima entre G. labiatus e G. lacustris da HBT do que entre as populações de G. labiatus da HBP e HBT. Em particular, a área do lábio teve uma grande plasticidade intraespecífica, não sendo informativa para diagnosticar G. lacustris de G. labiatus. Estimativas baseadas no relógio molecular sugeriram uma divergência recente entre as espécies da HBT (17.000 anos atrás), mas não entre as populações de G. labiatus da HBP e HBT (3,6 milhões de anos atrás). Finalmente, também foi encontrada uma linhagem genética de G. labiatus divergente no rio Camaquã, na HBP. Esses resultados mostram que a taxonomia atual de G. labiatus e G. lacustris não representa adequadamente as linhagens evolutivas nessas espécies.(AU)

A Century after! Rediscovery of the ancient catfish Diplomystes Bleeker 1858 (Siluriformes: Diplomystidae) in coastal river basins of Chile and its implications for conservation

The ancient catfish family Diplomystidae, with seven species endemic to rivers of southern South America, represents one of the oldest branches of the diverse order Siluriformes. With most species endangered, new reports of these species become extremely valuable for conservation. Currently, it is assumed that Diplomystes species inhabit only Andean (large) basins, and that they are extinct from coastal (small) basins from which their presence have not been recorded since 1919. Here, we document new records of the family Diplomystidae in the Laraquete and Carampangue basins, two coastal basins from the Nahuelbuta Coast Range, Chile, with no previous reports. This finding represents the rediscovery of the genus in coastal basins in more than a Century. Based on analysis of mitochondrial DNA sequences, the collected specimens were found to be closely related to Diplomystes nahuelbutaensis from the Andean Biobío Basin, but sufficiently differentiated to suggest that coastal basin populations are a different management unit. These populations are important because, contrary to previous thoughts, they prove these catfish can survive in small river networks, providing unique opportunities for research and conservation. The conservation category of Critically Endangered (CE) is recommended for the populations from the Laraquete and Carampangue basins.(AU)

La familia de bagres Diplomystidae, con siete especies endémicas de ríos del sur de Sudamérica, es uno de los linajes mas antiguos del diverso orden Siluriformes. Al estar la mayoría de las especies amenazadas, nuevos registros de éstas son extremadamente valiosos para su conservación. Actualmente, se ha asumido que los Diplomystidos se distribuyen solo en cuencas Andinas (más grandes), y que sus especies estarían extintas en cuencas de menor tamaño como las costeras, sin registros desde 1919. En este trabajo documentamos la familia Diplomistidae en las cuencas de Carampangue y Laraquete, dos cuencas costeras de la Cordillera de Nahuelbuta, Chile, lo que representa el primer registro de esta familia en estas cuencas costeras. Además, este hallazgo representa el re-descubrimiento de la familia en cuencas costeras después de un siglo. Sobre la base de análisis de ADN mitocondrial, los especímenes colectados se relacionaron más cercanamente con poblaciones de la especie Diplomystes nahuelbutaensis presente en la cuenca del Biobío. Sin embargo, existen diferencias genéticas suficientes entre las poblaciones costeras y las del Biobío para justificar su separación como unidad de manejo distinta. Estas poblaciones costeras son importantes porque demuestran que los Diplomístidos pueden sobrevivir en cuencas de pequeño tamaño, ofreciendo oportunidades únicas para su investigación y conservación. Se recomienda la categoría de conservación En Peligro Critico de Extinción (CR) para las poblaciones de las cuencas Laraquete y Carampangue.(AU)

Multiloci analyses suggest synonymy among Rhomboplites, Ocyurus and Lutjanus and reveal the phylogenetic position of Lutjanus alexandrei (Lutjanidae: Perciformes)

Lutjanidae comprises 21 genera and 135 species widespread throughout Atlantic, Indian and Pacific oceans. Nonetheless, the phylogenetic relationships of Lutjaninae remain uncertain. Furthermore, phylogenetic hypotheses for Lutjanus alexandrei, an endemic species from northeastern Brazilian coast, in Lutjanidae are absent so far. Therefore, we carried out multiloci analyses, combining both mitochondrial and nuclear DNA sequences in Lutjaninae species from Western Atlantic focusing on the controversial relationships among Lutjanus, Rhomboplites, and Ocyurus. Besides, we determined the phylogenetic position and dated the origin of L. alexandrei. The phylogenetics trees based on the 4.4 kb for 11 species corroborated the synonym among Lutjanus and the putative monotypic genera. For the dating of L. alexandrei, another nucleotide dataset (3.0 kb; 40 species) validated the genetic identity of this species that diverged from the sister taxon L. apodus between 2.5 - 6.5 Mya, probably as a result of the barrier caused by the muddy outflow from Orinoco and Amazon rivers along the coastal zone. This report is the most robust multiloci analysis to confirm the synonymy of the three genera of Lutjaninae from Western Atlantic and the first reliable inference about the phylogenetic relationships and origin of L. alexandrei.(AU)

A Família Lutjanidae compreende 21 gêneros e 135 espécies, distribuídas ao longo dos oceanos Atlântico, Índico e Pacífico. As relações filogenéticas dos Lutjaninae são incertas. Além disso, a espécie Lutjanus alexandrei, endêmica da costa nordeste do Brasil, não foi inclusa em nenhuma hipótese filogenética até o presente. Assim, realizamos uma análise integrando DNA mitocondrial e nuclear para espécies de Lutjaninae do Atlântico Ocidental, direcionada para a controversa relação entre Lutjanus, Rhomboplites e Ocyurus. Além disso, alocamos filogeneticamente L. alexandrei e datamos sua origem. As árvores filogenéticas baseadas em 4.4 kb de 11 espécies corroboraram a sinonímia entre os monotípicos e Lutjanus. Para a datação de L. alexandrei, outro banco de nuclueotídeos foi analisado (3.0 kb; 40 espécies), validando geneticamente a espécie e a colocando como irmã de L. apodus, da qual se separou entre 2.5 - 6.5 Mya, o que provavelmente foi provocado pela faixa enlameada na região costeira, influenciada pelas descargas dos rios Amazonas e Orinoco, que funciona como barreira. Este trabalho representa a mais robusta análise multiloci direcionada para a sinonimização dos três gêneros de Lutjaninae e a primeira hipótese filogenética a propor um posicionamento e origem para L. alexandrei.(AU)

Genetic variability of populations of Nyssomyia neivai in the Northern State of Paraná, Brazil

ABSTRACT The genetic study of sandfly populations needs to be further explored given the importance of these insects for public health. Were sequenced the NDH4 mitochondrial gene from populations of Nyssomyia neivai from Doutor Camargo, Lobato, Japira, and Porto Rico, municipalities in the State of Paraná, Brazil, to understand the genetic structure and gene flow. Eighty specimens of Ny. Neivai were sequenced, 20 from each municipality, and 269 base pairs were obtained. A total of 27 haplotypes and 28 polymorphic sites were found, along with a haplotypic diversity of 0.80696 and a nucleotide diversity of 0.00567. Haplotype H5, with 33 specimens, was the most common among the four populations. Only haplotypes H5 and H7 were present in all four populations. The population from Doutor Camargo showed the highest genetic diversity, and only this population shared haplotypes with those from the other municipalities. The highest number of haplotypes was sheared with Lobato which also had the highest number of unique haplotypes. This probably occurred because of constant anthropic changes that happened in the environment during the first half of the twentieth century, mainly after 1998. There was no significant correlation between genetic and geographical distances regarding these populations. However, the highest genetic and geographical distances, and the lowest gene flow were observed between Japira and Porto Rico. Geographical distance is a possible barrier between these municipalities through the blocking of haplotype sharing.

La mitocondria en la medicina legal y forense / The Mitochondrion in legal and forensic medicine

Las mitocondrias son las organelas intracelulares encargadas de suministrar la mayor parte de la energía necesaria para la actividad celular. Actúan, por lo tanto, como centrales energéticas de la célula y sintetizan ATP a expensas de los sustratos metabólicos. La intoxicación con ácido cianhídrico inhibe estos mecanismos y las alteraciones en el funcionamiento del metabolismo mitocondrial de origen genético o congénito producen innumerables patologías. El conocimiento de las patologías y disfunción de las mitocondrias es de importancia para realizar correctamente el diagnóstico de las causas de muerte. El ADN mitocondrial es de suma importancia en la medicina legal y forense para la identificación de las personas.

Mitochondria are organelles in the cell cytoplasm which supply most of the energy needed for cellular activity. They behave as cell’s power plants and synthesize ATP using metabolic substrates. Intoxication with Hydrocyanic Acid inhibits the synthesis of ATP, generating alterations in the mitochondrial metabolism, either genetic or congenital. The consequences of those alterations are innumerables pathologies. Understanding the pathologies and malfunctions of mitochondria help us to make the right diagnostic about the cause of death. In forensic medicine, mitochondrial DNA is of paramount relevance to people identification.

Molecular and morphological characterization of Contracaecum pelagicum (Nematoda) parasitizing Spheniscus magellanicus (Chordata) from Brazilian waters / Caracterização molecular e morfológica de Contracaecum pelagicum (Nematoda) parasito de Spheniscus magellanicus (Chordata) em águas brasileiras

Three new sequences of Mitochondrial cytochrome c-oxidase subunit 2 (mtDNA cox-2) from C. pelagicum parasite of Spheniscus magellanicus, the Magelanicus penguin, were determined from Brazilian waters. The sequences presented 99 and 98% of similarity with C. pelagicum sequences from Argentina, deposited on GenBank for the same genetic region and with a strong statistical support inferred from the phylogenetic tree. The morphological and ultrastructural studies that were carried out confirmed the genetic analysis.

Foram determinadas três novas sequências da região do Citocromo c-oxidase da subunidade II do DNA mitocondrial (cox-2 mtDNA) de Contracaecum pelagicum, parasito de Spheniscus magellanicus, pinguim Magalhães, de águas brasileiras. As sequências apresentaram 99 e 98% de similaridade com sequências de C. pelagicum da Argentina depositadas no GenBank da mesma região genética com forte suporte estatístico inferido pela arvore filogenética. Estudos morfológicos e ultraestruturais realizados confirmaram a identidade genética.

AS-PCR assay for 20 mtDNA SNP typing and haplotype frequency / 法医学杂志

OBJECTIVE@#To develop a multiplex allele-specific PCR (AS-PCR) assay with three-color fluorescence labeling for mitochondrial DNA (mtDNA) SNP typing.@*METHODS@#Based on the principle of AS-PCR, the primer sets were designed for 20 SNP located on the coding region of mtDNA and divided into 2 groups labeled with FAM and HEX fluorescence, respectively. A primer set included two forward (reverse) allelic specific primers with different sizes and a generic reverse (forward) primer. Blood samples from 200 unrelated individuals were analyzed by AS-PCR and capillary electrophoresis. Three random samples at least for each SNP site were examined and verified by direct sequencing. The haplotype frequency was investigated.@*RESULTS@#Distinct electropherograms of 200 blood samples were obtained successfully. The typing results of direct sequencing were identical to those obtained from AS-PCR. The minimum detectable DNA concentration was 0.2 pg under the system of 10 microL. The sensitivity of the DNA concentrations ranged from 0.5 to 5 pg. The 200 individuals were assigned into 15 haplotype, and the haplotype diversity was 0.906 0.@*CONCLUSION@#AS-PCR is a simple, rapid and efficient method for mtDNA SNP typing, and can be applied to forensic practice.

Especies de Anopheles presentes en el departamento del Putumayo y su infección natural con Plasmodium / Anopheles species present in the department of Putumayo and their natural infectivity with Plasmodium

Introducción. El departamento del Putumayo es una región endémica para malaria, o paludismo, causada principalmente por Plasmodium vivax . Los vectores en esta región incluyen Anopheles darlingi , el cual se ha encontrado solamente en el municipio de Puerto Leguízamo, y recientemente se incriminaron como vectores en Puerto Asís a las especies An. rangeli y An. oswaldoi . Objetivo. El propósito del trabajo fue determinar el papel de An. benarrochi B en la transmisión de malaria en este departamento, ya que se reporta como la especie más abundante que pica a los humanos. Materiales y métodos. Se recolectaron larvas y adultos de Anopheles spp. entre el 2006 y el 2008 en los municipios Puerto Leguízamo y Puerto Asís, y se obtuvieron secuencias del gen ITS-2 y del gen mitocondrial COI para confirmar las determinaciones taxonómicas por morfología. Se practicó la prueba ELISA para establecer la infección por P. vivax y P. falciparum. Resultados. Se identificaron 6.238 individuos correspondientes a 11 especies: An. albitarsis s.l. (1,83 %), An. benarrochi B (72,35 %), An. braziliensis (0,05 %), An. costai (0,06 %), An. darlingi (19,37 %), An. mattogrossensis (0,08 %), An. neomaculipalpus (0,13 %), An. oswaldoi s.l. (0,64 %), An. punctimacula (0,03 %), An. rangeli (5,12 %) y An. triannulatus s.l. (0,34 %). Se evaluaron 5.038 adultos por ELISA y 5 se encontraron positivos para P. vivax 210 y VK 247, todos pertenecientes a la especie An. benarrochi B. Conclusión. Los resultados sugieren que An. benarrochi B juega un papel en la transmisión de P. vivax en el departamento de Putumayo, dada su alta atracción por los humanos y su infección natural con Plasmodium spp.

Introduction: Putumayo is considered an endemic region for malaria transmission, mainly due to Plasmodium vivax. The vectors in this region are Anopheles darlingi , which has been found only in the municipality of Puerto Leguízamo, and An. rangeli and An. oswaldoi s.l. , which were recently incriminated as vectors in Puerto Asís. Objective: The purpose of this study was to determine the role of An. benarrochi B in malaria transmission in Putumayo, given that it is the most abundant species biting humans. Materials and methods: Collections of immature and adult stages of Anopheles spp. were made between 2006 and 2008 in the municipalities of Puerto Leguízamo and Puerto Asís in Putumayo, and sequences of internal transcribed spacer 2 ( ITS-2 ) of ribosomal DNA and the mitochondrial gene COI were obtained to confirm the morphological determinations. ELISA was carried out for P. vivax and P. falciparum infectivity. Results: A total of 6,238 specimens were identified, distributed in 11 species: An. albitarsis s.l. (1.83%), An. benarrochi B (72.35%), An. braziliensis (0.05%), An. costai (0.06%), An. darlingi (19.37%), An. mattogrossensis (0.08%), An. neomaculipalpus (0.13%), An. oswaldoi s.l. (0.64%), An. punctimacula (0.03%), An. rangeli (5.12%), and An. triannulatus s.l. (0.34%). A total of 5,038 adults were assessed by ELISA and 5 were found positive for P. vivax 210 and VK 247, all belonging to An. benarrochi B. Conclusion: The results suggest that An. benarrochi B plays a role in the transmission of P. vivax in Putumayo due to its high human contact and natural infection with Plasmodium sp.

Nail DNA and Possible Biomarkers: A Pilot Study / 예방의학회지

OBJECTIVES: Nail has been a substitute DNA source for genotyping. To investigate the integrity and consistency of nail DNA amplification for biomarker study, nail clippings from 12 subjects were collected at monthly intervals. The possibility of longer amplification and existence of GAPDH RNA/protein, were also investigated with three nail samples. METHODS: Three primer sets were designed for quantitative amplification of nuclear and mitochondrial genes and analysis of their consistency. The mean threshold cycles in amplification of the target genes were compared to test the consistency of polymerase chain reaction (PCR) performance among individual factors including age groups, sex, family, the nail source, and by the size of the amplification segments. RESULTS: The amplification of the target genes from nail DNA showed similar integrity and consistency between the nail sources, and among the serial collections. However, nail DNA from those in their forties showed earlier threshold cycles in amplification than those in their teens or seventies. Mitochondrial DNA (mtDNA) showed better DNA integrity and consistency in amplification of all three targets than did nuclear DNA (nucDNA). Over 9 kb of mtDNA was successfully amplified, and nested quantitative PCR showed reliable copy numbers (%) between the two loci. Reverse transcription PCR for mRNA and immunoblotting for GAPDH protein successfully reflected their corresponding amounts. Regarding the existence of RNA and protein in nails, more effective extraction and detection methods need to be set up to validate the feasibility in biomarker study. CONCLUSIONS: Nail DNA might be a feasible intra-individual monitoring biomarker. Considering integrity and consistency in target amplification, mtDNA would be a better target for biomarker research than nucDNA.

Asociación de niveles de lípidos y haplogrupos Amerindios de DNA mitocondrial en individuos chilenos hipercolesterolémicos tratados con atorvastatina / Association of serum lipid levels and mitochondrial DNA Amerindian haplogroups in hypercholesterolemic subjects receiving atorvastatin

Resumen: Introducción: La respuesta terapéutica a estatuías se ve influenciada por factores como la edad, género y etnicidad. Con respecto a esto, el background genético de la población chilena es predominantemente Amerindio, definido por la presencia de haplogrupos Amerindios A, B, C y D de DNA mitocondrial (mtDNA). Así, el objetivo del estudio fue evaluar la potencial asociación entre la presencia de haplogrupos Amerindios de mtDNA y niveles de lípidos en individuos chilenos hipercolesterolémicos tratados con Atorvastatina. Métodos: Un total de 42 individuos en dos centros de salud del sur de Chile fueron incluidos en el estudio. En el grupo de pacientes se evaluó la presencia de haplogrupos Amerindios de mtDNA por PCR-RFLP, además de la cuantificación de Colesterol Total, Triglicéridos, Colesterol-HDL y Colesterol-LDL, antes y después del tratamiento con Atorvastatina (10 mg/día). Resultados: El 88.1 por ciento de los sujetos presentó algún haplogrupo Amerindio, no observándose diferencias en los niveles de lípidos pre- tratamiento de acuerdo al haplogrupo. Interesantemente, individuos de haplogrupo B presentaron niveles mayores de Colesterol Total (B: 254 +/- 30 mg/dl v/s C: 213 +/- 48 mg/dl, D: 230 +/- 50 mg/dl; p= 0.0319) y Colesterol-LDL (B: 157 +/- 34 mg/dl v/s C: 118 +/- 45 mg/dl, D: 135 +/- 42 mg/dl; p=0.0344) post-tratamiento. Conclusiones: El haplogrupo B se asocia a niveles mayores de lípidos post-tratamiento en pacientes tratados con Atorvastatina. Estos hallazgos sugieren por primera vez, que la presencia de haplogrupo B de mtDNA determinaría una menor respuesta al tratamiento con Atorvastatina en individuos chilenos con background genético amerindio.

Background: Therapeutic response to statins is influenced by age, gender and ethnicity. The genetic background of the Chilean population is predominantly Amerindian, defined by the presence of mitochondrial DNA (mtDNA) Amerindian haplogroups A, B, C and D Amerindian haplogroups and serum lipid levéis in hypercholesterolemic Chilean subjects receiving atorvastatin Methods: 42 subjects from southern Chile were included. The presence of mtDNA Amerindian haplogroups was evaluated by PCR-RFLP; in addition, total cholesterol, triglycerides, HDL-cholesterol and LDL-cholesterol were measured before and after treatment with atorvastatin 10 mg/day. Aim: to evaluate a possible association of mtDNA. Ameridian haplogroups and serum lipid levels in hypercholesterolemic Chilean subjects receiving atorvastatin. Result: 88.1 percent of subjects exhibited some Amerindian haplogroup. No relation of lipid levels with haplogroups was observed before treatment. Interestingly, haplogroup B individuals had higher levels of total cholesterol compared to other haplogroups after treatment (haplogroup B : 254 +/- 30 mg/dl; C : 213 +/- 48 mg/dl; D : 230 +/- 50 mg/dl, p=0.0319). Corresponding levels for LDL-cholesterol after treatment in the three groups were 157 +/- 34,118 +/-45 and 135 +/-42 mg/ di, respectively, p=0.0344. Conclusion: Compared to other haplogroups, haplogroup B is associated to higher levels of lipids after treatment with atorvastatin. For the first time, these findings suggest that the presence of mtDNA haplogroup B determines a dimished response to atorvastatin in Chilean subjets with an Amerindian genetic background.

Mutational analysis of whole mitochondrial DNA in patients with MELAS and MERRF diseases

Mitochondrial diseases are clinically and genetically heterogeneous disorders, which make the exact diagnosis and classification difficult. The purpose of this study was to identify pathogenic mtDNA mutations in 61 Korean unrelated families (or isolated patients) with MELAS or MERRF. In particular, the mtDNA sequences were completely determined for 49 patients. From the mutational analysis of mtDNA obtained from blood, 5 confirmed pathogenic mutations were identified in 17 families, and 4 unreported pathogenically suspected mutations were identified in 4 families. The m.3243A>G in the tRNA(Leu(UUR)) was predominantly observed in 10 MELAS families, and followed by m.8344A>G in the tRNA(Lys) of 4 MERRF families. Most pathogenic mutations showed heteroplasmy, and the rates were considerably different within the familial members. Patients with a higher rate of mutations showed a tendency of having more severe clinical phenotypes, but not in all cases. This study will be helpful for the molecular diagnosis of mitochondrial diseases, as well as establishment of mtDNA database in Koreans.

Invalidación de autapomorfías putativas mediante análisis de filogenia molecular en Anopheles subgénero Nyssorhynchus / Invalidation of putative autapomorphic characters using molecular phylogenetic analyses in Anopheles subgenus Nyssorhynchus

Colectas e identificaciones realizadas en un área de transmisión malárica en el estado Sucre, Venezuela, mostraron mediante el uso de las claves ad hoc, la posible presencia en simpatría de poblaciones de Anopheles aquasalis Curry y An. benarrochi Gabaldón, Cova-García & López. Adicionalmente, se detectaron poblaciones de individuos que mostraron mezcla de los caracteres diagnósticos señalados en las claves. Debido a la importancia epidemiológica que representan ambas especies en dicha zona malárica, este estudio se orientó hacia la determinación de las tres entidades señaladas, mediante filogenia molecular con las secuencias de ADN mitocondrial (region Citocromo Oxidasa 1). En este sentido, el concepto de especie filogenética fue evaluada utilizando análisis de parsimonia máxima que mostraron politomías (nodos no resueltos) fuertemente apoyadas entre las secuencias inclusive la del haplotipo proveniente de un macho identificado como aquasalis, mostrando hipótesis (árboles) totales sin resolución ni apoyo de grupos internos con los tres morfotipos como un grupo natural único. Las distancias genéticas (Kimura-2P) mostraron que la variablidad inter-morfotipos están dentro de la varianza intra sugiriendo que los haplotipos problema son una única entidad polimórfica y con plasticidad fenotípica. Se concluye que An. aquasalis es la entidad única de las poblaciones estudiadas y que los caracteres morfológicos como: 1) vena Medial con escamas negras y 2) palpos maxilares con superficie ventral cubierta de escamas blancas, que son propuestas en la mayoría de las claves no son caracteres válidos para la identificación de hembras de An. benarrochi y que tales caracteres forman parte de la variación intraespecífica en An. aquasalis. Esta conclusión apoya la revisión de Faran (1980), quién no propone caracteres diferenciales para hembras de ambas especies, siendo indistinguibles por medio de los caracteres morfológicos tradicionalmente utilizados.

Collected specimens from a malaric area in Sucre State, Venezuela, using the ad hoc Keys, were identified as Anopheles aquasalis Curry and An. benarrochi Gabaldón, Cova-García & López populations. Additionally, other individuals have shown a mixture of diagnostic characters of both species. As a consequence of the epidemiological importance that both species represent in this malaric area and its correct identification for vector control aims, we addressed the research to the determination of both entities by mean of phylogenetic methods using mitochondrial DNA (Cytochrome Oxidase 1) from these three entities. In order to evaluate the phylogenetic species concept, the Maximum Parsimony analyses showed strong supported politomies (unresolved nodes), yielded solutions with no supported groups correlated with two or three morphological entities including the male haplotype identified as An. aquasalis. The genetic distances (K-2P) showed that the variability inter-morphotype is in the variance intra, suggesting that the haplotypes belong to the three morphotypes and represent a unique polymorphic species with phenotypic plasticity. We conclude that An. aquasalis is a unique taxonomic entity in the populations studied and also that the morphological characters such as: 1) medial vein with black scales and 2) maxillary palpus with ventral surface covered with white scales, proposed in most of the keys used are not valid characters to distinguish females of An. benarrochi from An. aquasalis as these characters belong to the intra-specific variation of An. aquasalis. This conclusion supports the Faran (1980) key, which did not report any morphological characters, showing undistinguished females under classical characters used

Populations analysis of the Brazilian Sharpnose Shark Rhizoprionodon lalandii (Chondrichthyes: Carcharhinidae) on the São Paulo coast, Southern Brazil: inferences from mt DNA sequences

Sharks of the genus Rhizoprionodon can be considered some of the most important predators along the trophic coastal marine ecosystems and represent an important economic resource for the small-scale fisheries, especially on the Brazilian coastline. In order to analyze the population structure of the shark Rhizoprionodon lalandii of São Paulo, Southeastern coast of Brazil, levels of genetic diversity were identified by nucleotide sequence analyses of the mitochondrial DNA control region. The results obtained from this study present moderate values of haplotype diversity and low nucleotide diversity. Although the AMOVA tests (ΦST = 0.08394, P < 0.01) had shown slightly differences among the studied samples, evidence for the occurrence of population structuring was not found, which may be a general feature of sharks living in coastal areas.

Tubarões do gênero Rhizoprionodon são considerados predadores de grande importância ao longo da cadeia trófica nos ecossistemas costeiros e marinhos, também representando um importante recurso econômico para a pesca, especialmente no litoral brasileiro. A fim de analisar a estrutura populacional do tubarão Rhizoprionodon lalandii no litoral de São Paulo, sudeste do Brasil, foram identificados os níveis de diversidade genética a partir da análise de sequências nucleotídicas da região controladora do DNA mitocondrial. Os dados obtidos neste estudo apresentam valores moderados de diversidade haplotípica e baixos índices de diversidade nucleotídica. Embora os testes de AMOVA (ΦST = 0,08394, P < 0,01) tenham revelado uma pequena diferença entre as amostras estudadas, evidências sobre a ocorrência de estruturação populacional não foram encontradas o que pode representar uma característica geral para tubarões vivendo em áreas costeiras.

Identificación de especies en productos de origen animal mediante PCR / PCR-species-identication in animal products

La identificación de la especie en productos de origen animal (carne, leche o sus derivados) se hace necesaria y de exigencia por los consumidores modernos, entre otras razones: i) para evitar fraude económico, ya sea por sustitución o adulteración del mismo, ii) por motivos de salud humana, tales como alergias alimentarías, iii) por implicaciones culturales; de allí que se debe contar con herramientas analíticas y sensibles para dicha identificación tales como el análisis de fragmentos de ADN en especial de origen mitocondrial (gen 12S ARNr) dada su particularidad de ser especifica de especies. A tal fin se estableció una metodología de identificación mediante la amplificación de fragmentos específicos de ADN mitocondrial (ADNm) a partir de muestras biológicas de las principales especies animales implicadas en la producción de carnes o alimentos (bovina, porcina, ovina, caprina, equina, asnos, felina y canina), específicamente de una fracción parcial del gen 12sARNr de una región conservada usando unos cebadores comunes para dichas especies, un “reverse” especifico de especie y análisis posterior mediante geles de agarosa al 1,5 por ciento y amplificación de fragmentos que oscilaron entre 150 y 364 pb. Los resultados indican que se puede identificar la especie a la que pertenece la muestra analizada en el 100 por ciento de los casos, ofreciendo una herramienta especifica para determinar la especie en alimentos de origen animal.

Nowadays identification of animal-origin products (meat, milk and dairy products) is of paramount importance to costumers and specially as for species identification for a number of reasons: i) to avoid economic fraud for substitution or alteration of the product ; ii) to avoid health issues such as food allergies; iii) for culturals reasons. There for the value of analytic and sensitive identification tools such as DNA fragments analysis, especially those of mitochondrial origin (12S rRNA gene) because of its species-specific character. An identification methodology through amplification of a species-conserved region of 12S rRNA gene (forward primer) and of a species-specific region of the same gene (reverse primer). Template DNA was extracted from biological samples of bovine, swine, ovine, caprine, equine and canine origin. After 1.5 percent agarose gel electrophoresis, fragments ranging from 150 to 364 bp were observed. Results show that species could be easily identified through PCR in all cases and that this methodology could be a specific tool for determining the origin of animal products.

DNA barcoding of Brazilian sea turtles (Testudines)

Five out of the seven recognized species of sea turtles (Testudines) occur on the Brazilian coast. The Barcode Initiative is an effort to undertake a molecular inventory of Earth biodiversity. Cytochrome Oxidase c subunit I (COI) molecular tags for sea turtle species have not yet been described. In this study, COI sequences for the five species of sea turtles that occur in Brazil were generated. These presented widely divergent haplotypes. All observed values were on the same range as those already described for other animal groups: the overall mean distance was 8.2 percent, the mean distance between families (Dermochelyidae and Cheloniidae) 11.7 percent, the mean intraspecific divergence 0.34 percent, and the mean distance within Cheloniidae 6.4 percent, this being 19-fold higher than the mean divergence observed within species. We obtained species-specific COI barcode tags that can be used for identifying each of the marine turtle species studied.

Green turtles (Chelonia mydas) foraging at Arvoredo Island in Southern Brazil: genetic characterization and mixed stock analysis through mtDNA control region haplotypes

We analyzed mtDNA control region sequences of green turtles (Chelonia mydas) from Arvoredo Island, a foraging ground in southern Brazil, and identified eight haplotypes. Of these, CM-A8 (64 percent) and CM-A5 (22 percent) were dominant, the remainder presenting low frequencies (< 5 percent). Haplotype (h) and nucleotide (n) diversities were 0.5570 ± 0.0697 and 0.0021 ± 0.0016, respectively. Exact tests of differentiation and AMOVA Fi ST pairwise values between the study area and eight other Atlantic foraging grounds revealed significant differences in most areas, except Ubatuba and Rocas/Noronha, in Brazil (p > 0.05). Mixed Stock Analysis, incorporating eleven Atlantic and one Mediterranean rookery as possible sources of individuals, indicated Ascension and Aves islands as the main contributing stocks to the Arvoredo aggregation (68.01 percent and 22.96 percent, respectively). These results demonstrate the extensive relationships between Arvoredo Island and other Atlantic foraging and breeding areas. Such an understanding provides a framework for establishing adequate management and conservation strategies for this endangered species.

Genetic characterisation of populations of the critically endangered Goliath grouper (Epinephelus itajara, Serranidae) from the Northern Brazilian coast through analyses of mtDNA

The Goliath grouper (Epinephelus itajara) is one of the most endangered species of fish of the subfamily Epinephelinae. Slow to develop and mature, and dependent on mangrove habitats for breeding, the species also suffers intense harvesting, which has reduced drastically in numbers in many areas. To contribute to the understanding of the characteristics of E. itajara populations, we conducted a molecular genetics study of the species, focusing on populations from the Northern Brazilian coast. The mtDNA control region (D-loop) of 116 individuals from five localities (Bragança, Ajuruteua, Parnaíba, Fortaleza and Natal) was analysed, and a sequence of 499 base pairs identified. Analyses of the sequences indicated that genetic variability was generally lower in E. itajara than in other endangered species of the genus. AMOVA found no significant grouping structure among the populations. Nested Clade Analysis revealed a significant association between genetic variability and geographic distribution among only three populations (Ajuruteua, Parnaíba and Natal). Genetic diversity was higher in populations from the Amazon region, which may be related to the better conservation of mangrove habitats in this area. Therefore, the present study could be used for the implementation of conservation and management measures in order to protect and consolidate these populations.

Análisis de ADN mitocondrial en una muestra de restos óseos arcaicos del periodo Herrera en la sabana de Bogotá / Mitochondrial DNA analysis on pre-Columbian bone remains of the Herrera period

Introducción. Los restos óseos arcaicos son fuente privilegiada de información biológica y su caracterización genética permite confirmar o descartar filiaciones propuestas por otras aproximaciones científicas. La historia precolombina de los Andes orientales se divide en tres periodos principales: I) un poblamiento temprano por parte de grupos cazadores-recolectores; II) un periodo intermedio (Herrera) de pueblos con agricultura incipiente, y III) un periodo tardío de pueblos chibchas, agrícolas y alfareros (agroalfarero). Objetivo. Analizar el ADN mitocondrial de restos óseos del periodo Herrera. Materiales y métodos. Se analizaron 11 individuos pertenecientes al yacimiento arqueológico Madrid 2-41, con una edad aproximada de 2.000 años. Un fragmento (192 pb) del segmento hipervariable I fue amplificado y secuenciado, siguiendo criterios estrictos de autenticidad deADN arcaico. Las secuencias se compararon con las existentes en bases de datos de Norteamérica y Europa usando herramientas bioinformáticas. Resultados. Todas las secuencias resultaron idénticas y fueron clasificadas como haplogrupo B. Esto puede relacionarse con el tipo de entierro ritual practicado en Madrid 2-41, es decir, probablemente los individuos analizados hagan parte de una familia jerárquicamente importanteen la antigua sociedad Herrera. La búsqueda de secuencias homólogas en las bases de datos estadounidense y europea no arrojó coincidencias exactas, aunque existe el reporte de un individuo amazónico de ~4.000 años de antigüedad (Brasil) cuya secuencia coincide con la hallada en Madrid 2-41.Conclusión. Los individuos del yacimiento arqueológico Madrid 2-41 están estrechamente emparentados entre sí por línea materna y presentan una secuencia aparentemente ausente en poblaciones actuales.

Optimization of methods to assess mitochondrial DNA in archival paraffin-embedded tissues from mammary canine tumors / Otimização dos métodos para avaliar o DNA mitocondrial obtido a partir de tumores mamários caninos incluídos em parafina

In this study we describe the alterations used to extract and amplify mitochondrial desoxyribonucleic acid (DNA) from formalin-fixed paraffin-embedded samples of canine mammary tumors. The epithelial and mesenchymal components (chondromyxoid and chondroid) of each tumor, as well as the normal mammary gland tissues, were manually microdissected from 19 mixed canine mammary tumors (10 benign mixed tumors and nine carcinomas arising in mixed tumors). DNA was extracted by Invisorb® Spin Tissue Mini Kit, with protocol changes proposed by the manufacturer. A 273-bp fragment was amplified by polymerase chain reaction (PCR) and submitted to automatic sequence analysis. The fragment was successfully analyzed in 100 percent of the samples. However, an additional lysis step, the reduction of volume in buffer solutions and PCR, a higher annealing temperature and an increase in the number of PCR cycles were required. The initial PCR products were diluted and re-amplified in six samples so that they could be successfully analyzed.

A presente comunicação descreve as modificações usadas para extrair e amplificar o DNA mitocondrial obtido de amostras de tumores mamários caninos fixados em formol tamponado a 10 por cento e incluídos em parafina. Os componentes epiteliais e mesenquimais (condromixóide e condróide), bem como a mama normal adjacente, foram microdissectados manualmente de 19 tumores mamários (10 tumores mistos benignos e nove carcinomas em tumores mistos). O DNA foi extraído utilizando-se o Invisorb® Spin Tissue Mini Kit com modificações do protocolo proposto pelo fabricante. Um fragmento de 273-pb foi amplificado por reação em cadeia da polimerase (PCR) e seqüenciado em seqüenciador automático. O fragmento foi analisado em 100 por cento das amostras, entretanto modificações como lise adicional, redução do volume das soluções de extração e PCR, aumento da temperatura de anelamento e do número de ciclos de amplificação foram necessárias. Em seis amostras os produtos iniciais de PCR foram diluídos e reamplificados para obtenção de sucesso.

Fine scale gene flow and individual movements among subpopulations of Centrolene prosoblepon (Anura: Centrolenidae)

Dispersal capabilities determine and maintain local gene flow, and this has implications for population persistence and/or recolonization following environmental perturbations (natural or anthropogenic), disease outbreaks, or other demographic collapses. To predict recolonization and understand dispersal capacity in a stream-breeding frog, we examined individual movement patterns and gene flow among four subpopulations of the Neotropical glassfrog, Centrolene prosoblepon, at a mid-elevation cloud forest site at El Copé, Panama. We measured male movement directly during a two year mark-recapture study, and indirectly with gene flow estimates from mitochondrial DNA sequences (mtDNA). Individuals of this species showed strong site fidelity: over two years, male frogs in all four headwater streams moved very little (mean = 2.33 m; mode = 0 m). Nine individuals changed streams within one or two years, moving 675-1,108 m. For those males moving more than 10 m, movement was biased upstream (p < 0.001). Using mtDNA ND1 gene sequences, we quantified gene flow within and among headwater streams at two spatial scales: among headwater streams within two adjacent watersheds (2.5 km2) and among streams within a longitudinal gradient covering 5.0 km2. We found high gene flow among headwater streams (phi(ST) = 0.007, p = 0.325) but gene flow was more limited across greater distances (phi(CT) = 0.322, p = 0.065), even within the same drainage network. Lowland populations of C. prosoblepon potentially act as an important source of colonists for upland populations in this watershed.

La capacidad de dispersión determina y mantiene el flujo genético local, y esto tiene implicaciones para la persistencia poblacional y/o la recolonización que sigue a perturbaciones ambientales. Examinamos patrones individuales de movimiento y flujo genético entre subpoblaciones de Centrolene prosoblepon (Anura: Centrolenidae) en un sitio de elevación media en El Copé, Panamá. Medimos directamente el movimiento de los machos durante un estudio de marcado-recaptura, e indirectamente con estimaciones de flujo genético a partir de secuencias de ADN mitocondrial (mtDNA). Los individuos mostraron fuerte fidelidad a su lugar: por más de dos años, las ranas macho de los cuatro arroyos al inicio del río se movieron muy poco (promedio = 2.33 m; moda = 0 m). Nueve individuos cambiaron de corriente de agua en uno o dos años, moviéndose 675-1 108 m. Usando la secuencia genética ND1 del ADN mitocondrial, medimos el flujo genético en dos escalas espaciales: entre arroyos que originan el río (2.5 km2) y entre arroyos con un gradiente longitudinal en 5.0 km2. Encontramos un flujo genético alto entre los arroyos al inicio del río (f = 0.007, p = 0.325 y otro más limitado en distancias mayores (f = 0.322, p = 0.065).