DNA barcoding revels first records of three rare coleopteran genera in Northern lakes of Egypt / O código de barras de DNA revela os primeiros registros de três gêneros raros de coleópteros nos lagos do norte do Egito

Abstract One aquatic coleopteran species from family Dytiscidae and two aquatic coleopteran genera from family Hydrophilidae were recorded in the summer period and represent first records in the Egyptian lakes. Beetles were collected from two northern lakes, Lake Idku and Lake Burullus. They were identified by morphological characteristics as well as the mtDNA barcoding method. A molecular phylogenetic approach was used to determine the genetic identity of the collected samples based on the mitochondrial cytochrome oxidase I (COI). Prodaticus servillianus (Dytiscidae) from Egypt showed no significant difference in the COI region and they are highly similar to P. servillianus from Madagascar. The phylogenetic analysis revealed that the other two coleopteran genera belong to family Hydrophilidae. Based on COI only, there is no clear evidence for their genetic identity at the species level. So, we defined them to the closest taxon and denoted them as Cymbiodyta type A and B. The results indicated that resolving the molecular identity of the aquatic beetles from northern lakes of Egypt need more considerations in the field of biological conservation. We concluded that utilization of COI as a barcoding region for identifying some coleopteran species is not sufficient and additional molecular markers are required to uncover the molecular taxonomy at deep levels.

Resumo Uma espécie de coleópteros aquático da família Dytiscidae e dois gêneros de coleópteros aquáticos da família Hydrophilidae foram registrados no período de verão e representam os primeiros registros nos lagos egípcios. Os besouros foram coletados em dois lagos do norte, o lago Idku e o lago Burullus, e identificados por características morfológicas e pelo método de código de barras mtDNA. Uma abordagem filogenética molecular foi usada para determinar a identidade genética das amostras coletadas com base no citocromo oxidase I mitocondrial (COI). Prodaticus servillianus (Dytiscidae) do Egito não mostrou diferença significativa na região COI e é altamente semelhante a P. servillianus de Madagascar. A análise filogenética revelou que os outros dois gêneros de coleópteros pertencem à família Hydrophilidae. Com base apenas no COI, não há evidências claras de sua identidade genética no nível da espécie. Assim, nós os agrupamos no táxon mais próximo e os denominamos Cymbiodyta tipo A e B. Os resultados indicaram que a identidade molecular dos besouros aquáticos dos lagos do norte do Egito precisa de mais considerações no campo da conservação biológica. Concluímos que a utilização de COI como região de código de barras para identificar algumas espécies de coleópteros não é suficiente, sendo necessários marcadores moleculares adicionais para descobrir a taxonomia molecular em níveis profundos.

Accediendo al pasado: uso de especímenes de colección como fuentes de información genética para el género Bombus (Hymenoptera: Apidae)

Introducción: Recientemente ha tomado relevancia el uso de especímenes de museo como fuente de información genética para desarrollar estudios que resuelven preguntas taxonómicas, ecológicas, demográficas y evolutivas a diversas escalas temporales y geográficas. Sin embargo, material genético obtenido a partir de ejemplares depositados en colecciones biológicas es poco usado, debido al deterioro natural del ADN preservado en dichos ejemplares, de manera que la obtención de material genético de calidad es demandante en términos de tiempo y dinero. Objetivo: Usando material de museo, identificar una secuencia mini-barcode que pueda ser empleada en la determinación taxonómica, y que a su vez suministre información que permita la estimación de relaciones filogenéticas de especies del género Bombus. Métodos: Se estandarizó el protocolo de extracción de ADN a partir de la extremidad mesotoracica derecha y/o una muestra de músculo torácico de 96 especímenes depositados en la colección LABUN entre 7 y 38 años atrás. Las diferentes combinaciones de oligonucleótidos evaluadas permitieron amplificar fragmentos de 152 a 407 pares de bases (pb) del gen mitocondrial Cytochrome Oxidase I (COI). Usando como plantilla un grupo de 31 secuencias amplificadas a partir de especímenes recolectados recientemente, los fragmentos obtenidos de los especímenes del museo fueron ensamblados y analizados en un marco filogenético. Además, se realizó un análisis de red de haplotipos para evaluar en detalle las relaciones entre los haplotipos mitocondriales resultantes. Resultados: Se determinó un mayor éxito de extracción de ADN a partir de muestras de extremidad depositadas a partir del año 1982.Entretanto, la amplificación exitosa de fragmentos de más de 300 pares de bases (pb) se logró principalmente en muestras depositadas en fechas posteriores a 1999, lo que indica una mayor integridad del material genético recuperado de individuos de 19 años de recolección en adelante. Aunque todos los fragmentos evaluados pueden ser empleados como mini-barcode, solo con uno se obtiene una topología similar a la observada con el fragmento completo. Se detectó una gran variacion genética, particularmente al interior de las especies Bombus atratus y B. funebris, en las que se reveló una clara estructura filogeográfica. Conclusiones: Se obtuvieron nuevas secuencias de códigos de barras mediante extracción de ADN y protocolo de amplificación de muestras de museos. Además, se generó nueva información sobre la variabilidad genética intraespecífica, detectando la presencia de haplotipos mitocondriales únicos que podrían constituir Unidades Significativas Evolutivas sujetas a conservación. Dicha información es de vital importancia para formular estrategias de conservación para estos polinizadores en Colombia.

Introduction: The use of museum specimens as a source of genetic information to develop studies that resolve taxonomic, ecological, demographic, and evolutionary questions at various temporal and geographic scales, has recently become relevant. However, genetic material obtained from specimens deposited in biological collections is not used frequently due to the natural deterioration of the DNA preserved in these specimens. Getting quality genetic material is demanding in terms of time and money. Objective: By using museum material,to identify a mini-barcode sequence that can be used in the taxonomic determination and provides information that allows the estimation of phylogenetic relationships of species of the genus Bombus. Methods: The DNA extraction protocol for museum samples was standardized using the mesothoracic right leg and / or a sample of thoracic muscle of 96 specimens deposited in the LABUN collection between 7 and 38 years ago. Different combinations of oligonucleotides allowed to amplify fragments from 152 to 407 base pairs (bp) of the mitochondrial gene Cytochrome Oxidase I (COI). Using as a template a group of 31 sequences amplified from recently collected specimens, the fragments obtained from the museum specimens were assembled and analyzed in a phylogenetic framework. Additionally, a haplotype network analysis was performed in order to evaluate in detail the relationships between the resulting mitochondrial haplotypes. Results: The greatest success of DNA extraction was achieved from limb samples deposited since the year 1982 on. Meanwhile, successful amplification of fragments longer than 300 base pairs (bp) was achieved mostly in samples deposited on dates after 1999, which indicates greater integrity of the genetic material recovered from individuals of 19 years of collection and onwards. Although all the fragments evaluated can be used as mini-barcode, only with one primer pair, it was possible to obtain a topology similar to that observed with the complete fragment. A large genetic variation was detected, particularly within the Bombus atratus and B. funebris species, in which a clear phylogeographic structure was revealed. Conclusions: New barcode sequences were obtained through DNA extraction and amplification protocol from museum samples. Furthermore, new information on intraspecific genetic variability was generated, detecting the presence of unique mitochondrial haplotypes that could constitute management units subject of conservation. Such information is of vital importance to formulate conservation strategies for these pollinators in Colombia.

Molecular phylogeny and evolution of Pulmonata (Mollusca: Gastropoda) on the basis of mitochondrial (16S, COI) and nuclear markers (18S, 28S): an overview

The phylogenetic relationships among the major groups of Pulmonata were studied by the information derived from a concatenated dataset consisting of mitochondrial (16S and COI) and nuclear (18S and 28S) markers. Heterobranchia are recovered as monophyletic. Euthyneura as paraphyletic due to the emergence of taxa from Opisthobranchia and lower Heterobranchia. The major groups of Pulmonata, namely Stylommatophora, Veronicellidae, Onchidiidae, Otinoidea, Siphonarioidea and Hygrophila are recovered as monophyletic. Monophyly of Basommatophora was not confirmed due to the variable position of Siphonarioidea and Amphiboloidea. Evolutionary divergence times for different taxa were also estimated using a relaxed molecular clock method in Bayesian evolutionary analysis by sampling trees (BEAST). The common ancestor of Heterobranchia and Caenogastropoda was originated in the Silurian period and the common ancestors of Euthyneura and Pulmonata were originated in the Carboniferous and lower Triassic periods, respectively.

Genetic diversity of Echinococcus vogeli in the western Brazilian Amazon

Human polycystic echinococcosis is a parasitic infection caused by the larval stage of Echinococcus vogeli, which occurs in rural areas of Central and South America. Until now, little information on the genetic variability of E. vogeli is available. Here, 32 samples from human-excised E. vogeli cysts had a 396-bp sequence of the mitochondrial cytochrome oxidase I (COI) gene sequenced and compared to another 17 COI sequences representing nine Echinococcus species. A Bayesian COI tree revealed that all E. vogeli sequences formed a monophyletic and well-supported clade with an E. vogeli reference sequence. The occurrence of geographically restricted E. vogeli COI haplotypes suggests retention of ancestral polymorphisms with little migration in Acre, Brazil.

DNA barcoding revels first records of three rare coleopteran genera in Northern lakes of Egypt

Abstract One aquatic coleopteran species from family Dytiscidae and two aquatic coleopteran genera from family Hydrophilidae were recorded in the summer period and represent first records in the Egyptian lakes. Beetles were collected from two northern lakes, Lake Idku and Lake Burullus. They were identified by morphological characteristics as well as the mtDNA barcoding method. A molecular phylogenetic approach was used to determine the genetic identity of the collected samples based on the mitochondrial cytochrome oxidase I (COI). Prodaticus servillianus (Dytiscidae) from Egypt showed no significant difference in the COI region and they are highly similar to P. servillianus from Madagascar. The phylogenetic analysis revealed that the other two coleopteran genera belong to family Hydrophilidae. Based on COI only, there is no clear evidence for their genetic identity at the species level. So, we defined them to the closest taxon and denoted them as Cymbiodyta type A and B. The results indicated that resolving the molecular identity of the aquatic beetles from northern lakes of Egypt need more considerations in the field of biological conservation. We concluded that utilization of COI as a barcoding region for identifying some coleopteran species is not sufficient and additional molecular markers are required to uncover the molecular taxonomy at deep levels.

Resumo Uma espécie de coleópteros aquático da família Dytiscidae e dois gêneros de coleópteros aquáticos da família Hydrophilidae foram registrados no período de verão e representam os primeiros registros nos lagos egípcios. Os besouros foram coletados em dois lagos do norte, o lago Idku e o lago Burullus, e identificados por características morfológicas e pelo método de código de barras mtDNA. Uma abordagem filogenética molecular foi usada para determinar a identidade genética das amostras coletadas com base no citocromo oxidase I mitocondrial (COI). Prodaticus servillianus (Dytiscidae) do Egito não mostrou diferença significativa na região COI e é altamente semelhante a P. servillianus de Madagascar. A análise filogenética revelou que os outros dois gêneros de coleópteros pertencem à família Hydrophilidae. Com base apenas no COI, não há evidências claras de sua identidade genética no nível da espécie. Assim, nós os agrupamos no táxon mais próximo e os denominamos Cymbiodyta tipo A e B. Os resultados indicaram que a identidade molecular dos besouros aquáticos dos lagos do norte do Egito precisa de mais considerações no campo da conservação biológica. Concluímos que a utilização de COI como região de código de barras para identificar algumas espécies de coleópteros não é suficiente, sendo necessários marcadores moleculares adicionais para descobrir a taxonomia molecular em níveis profundos.

Taxonomía molecular de Anopheles del Ecuador mediante ADN mitocondrial (Citocromo c Oxidasa I) y optimización por Parsimonia Máxima / Molecular Taxonomy of Anopheles from Ecuador, using mitochondrial DNA (Cytochrome c Oxidase I) and Maximum Parsimony optimization

La identificación de especies de Anopheles es compleja debido a la presencia de varios complejos de especies o especies cripticas cuyos ejemplares son de difícil distinción morfológica. Se corroboró o se corrigió la identificación de especies de Anopheles spp. capturadas en Ecuador, utilizando 393 secuencias de ADN mitocondrial, Citocromo Oxidasa I (COI) de 36 especies de Anofelinos neotropicales depositadas en GenBank y mediante la construcción de árboles filogenéticos usando parsimonia máxima. Se analizaron las identificaciones moleculares de cinco especies de Anopheles mediante los criterios de monofília y topología del árbol, secuencias provenientes de la localidad tipo y la divergencia genética intra/inter especies. Las topologías de los árboles resultantes corroboran la identificación de secuencias/especies de Anopheles (Anopheles) pseudopunctipennis Theobald, An. (Ano.) eiseni Coquillett y An. (Nyssorhynchus) albimanus Wiedemann; se corrige la identificación molecular de An. (Nys.) rangeli Gabaldón, Cova-García y López (no oswaldoi) y permanece en duda la identificación correcta de las secuencias relacionadas el grupo Punctimacula, hasta tener disponibles un mayor número de secuencias COI de especies relacionadas. La matriz de ADN-COI de 241 secuencias/haplotiposx36 especies construida para estos análisis resultará de utilidad para la identificación molecular de especies de Anofelinos de Ecuador y del Neotrópico con base en la porción de 520 pb entre 2.272 a 2.792pb de COI.

The Anopheles species identification is complicated by the presence of several complexes of species and species whose specimens are difficult morphological distinction. We use 393 mitochondrial DNA sequences, Cytochrome Oxidase I (COI) of 36 Neotropical species of Anophelinae deposited in GenBank and by constructing phylogenetic trees using maximum parsimony the objective was to corroborate or correct the molecular identification of five sequences/species of Anopheles collected in Ecuador and also availables in Genbank. We identified the taxonomic units, based on monophyly, phylogenetic species definition, tree topology, ocurrence of sequences from the type locality and genetic intra/inter divergence of clades. The topologies of the resulting trees corroborate the identification of sequences of Anopheles (Anopheles) pseudopunctipennis Theobald, An. (Ano.) eiseni Coquillett y An. (Nyssorhynchus) albimanus Wiedemann, while the molecular identification of An. (Nys.) rangeli Gabaldón, Cova-García y López (not “oswaldoi”) is corrected and remains in doubt the correct identification of related sequences for sequences belonging to Punctimacula group, until have available a greater number of COI sequences from related species. However according to the criteria of type locality, these are not punctimacula in sensu stricto. Finally, the matrix of alignment DNA-COI haplotypes sequences of 241x36 species built for these analyzes will be useful for molecular identification of Anophelinae species from Ecuador and the Neotropics based on the portion of 520 bp COI (between 2,272- 2,792 bp).

Estrutura populacional e filogeografia de Panulirus argus (Latreille, 1804) / Population structure and phylogeography of Panulirus argus (Latreille, 1804)

Panulirus argus (Latreille, 1804) é uma das principais espécies de lagosta no Atlântico, sendo um dos maiores recursos pesqueiros do Atlântico Ocidental, onde apresenta um alto valor comercial. A forte explotação da espécie resulta em uma grande pressão sobre suas populações. Recentemente, foi descoberto que sob o binômio P. argus estão contidas duas espécies crípticas que ocorrem em alopatria, uma na região do Caribe e outra na costa brasileira. Esta tese tem como objetivo estudar como se estruturam geneticamente as populações dessas duas espécies, com o propósito de fornecer mais informações para a determinação de estoques e um correto manejo das espécies, e analisar os processos históricos evolutivos que moldaram suas histórias demográficas. Para tal, foram estudados dois marcadores mitocondriais (região controle e o gene da Citocromo Oxidase I) e loci de microssatélites de indivíduos de 7 regiões do Caribe (Florida, Bahamas, Turks e Caicos, Porto Rico, Cuba, Colômbia e Venezuela) e 11 estados do Brasil (Pará, Maranhão, Piauí, Ceará, Rio Grande do Norte, Pernambuco, Alagoas, Bahia, Espírito Santo, Rio de Janeiro e São Paulo). Dentro de cada espécie foram observadas duas linhagens mitocondriais diferentes, que co-ocorriam, de maneira homogênea, ao longo de suas distribuições. Hipotetiso que essas linhagens foram formadas a partir de um evento de vicariância com contato secundário ou como consequência de um efeito gargalo seguido de expansão. As duas linhagens são evidentes nas sequências da região controle mitocondrial, mas no gene da COI foram evidentes apenas em P. cf. argus do Caribe. As linhagens do Brasil se separaram há aproximadamente 233 - 288 mil anos e cada uma sofreu expansão em tempos diferentes, a primeira se expandiu há 100 mil anos e a segunda linhagem há 50 mil anos. As linhagens do Caribe se separaram cerca de 1 milhão de anos atrás e possuem o mesmo tempo de expansão, 50 mil anos. Os microssatélites não revelaram subdivisão populacional ...

Panulirus argus (Latreille, 1804) is one of the main lobster species in the Atlantic, and one of the largest fisheries in the western Atlantic, with a high commercial value. The heavy exploitation of the species results in much pressure on its populations. Recently, it was discovered that under the name P. argus there are two cryptic species that occur in allopatry, one in the Caribbean and the other on the coast of Brazil. This thesis studies the population genetic structure of those two species with the purpose of providing more information to delimitate stocks for fisheries management, and for understanding the historical processes that have shaped their evolutionary demographic histories. For this, we analysed two mitochondrial markers (control region and the Cytochrome Oxidase I gene) and microsatellite markers of individuals from 7 localities in the Caribbean (Florida, Bahamas, Turks and Caicos, Puerto Rico, Cuba, Colombia, and Venezuela) and 11 States of Brazil (Pará, Maranhão, Piauí, Ceará, Rio Grande do Norte, Pernambuco, Alagoas, Bahia, Espírito Santo, Rio de Janeiro, and São Paulo). Within each species two different mitochondrial lineages were observed. They occurred throughout their distributions, and it is hypothesized that they were formed from a vicariance event with secondary contact or are the result of a genetic bottleneck followed by expansion. The lineages of P. cf. argus from Brazil were only observed in the mitochondrial control region and were separated approximately 233-288 thousand years ago, and each lineage underwent expansion at different times: the first expanded 100,000 years ago and the second 50,000 years ago. The lineages of the Caribbean species were found for the two mitochondrial markers. They were separated about 1 million years ago and have had the same expansion time, 50,000 years. Microsatellites revealed no population subdivision for either species, but the molecular markers together suggest a differential gene...

A morphometric and molecular study of Anastrepha pickeli Lima (Diptera: Tephritidae)

This study investigated the level of morphometric and genetic variability among populations of Anastrepha pickeli Lima from several localities in Brazil, one locality in Bolivia and one in Paraguay. Traditional and geometric morphometric analyses were used, as well as sequencing of a fragment of the cytochrome oxidase gene (COI). Six variables were measured from the aculeus for traditional morphometric analysis and 14 landmarks from the right wing were used for geometric analysis, using 10 specimes/population. The aculeus tip length, aculeus width at the end of the cloaca opening, and the serrate part length contributed with 62.7 percent for grouping. According to the results from traditional morphometry, there was no significant difference, but the multivariate tests showed that the canonical variables were statistically significant, indicating a difference in the wing conformation among populations. Molecular phylogenetic analysis indicated that the populations clustered into three clades and revealed a high level of genetic variation within A. pickeli populations from various geographic regions. Anastrepha pickeli populations differed among them according to the methods used in this study, showing incongruence among the methods used.