A multi-locus approach to barcoding in the Anopheles strodei subgroup (Diptera: Culicidae)

Abstract Background The ability to successfully identify and incriminate pathogen vectors is fundamental to effective pathogen control and management. This task is confounded by the existence of cryptic species complexes. Molecular markers can offer a highly effective means of species identification in such complexes and are routinely employed in the study of medical entomology. Here we evaluate a multi-locus system for the identification of potential malaria vectors in the Anopheles strodei subgroup. Methods Larvae, pupae and adult mosquitoes (n = 61) from the An. strodei subgroup were collected from 21 localities in nine Brazilian states and sequenced for the COI, ITS2 and white gene. A Bayesian phylogenetic approach was used to describe the relationships in the Strodei Subgroup and the utility of COI and ITS2 barcodes was assessed using the neighbor joining tree and “best close match” approaches. Results Bayesian phylogenetic analysis of the COI, ITS2 and white gene found support for seven clades in the An. strodei subgroup. The COI and ITS2 barcodes were individually unsuccessful at resolving and identifying some species in the Subgroup. The COI barcode failed to resolve An. albertoi and An. strodei but successfully identified approximately 92% of all species queries, while the ITS2 barcode failed to resolve An. arthuri and successfully identified approximately 60% of all species queries. A multi-locus COI-ITS2 barcode, however, resolved all species in a neighbor joining tree and successfully identified all species queries using the “best close match” approach. Conclusions Our study corroborates the existence of An. albertoi, An. CP Form and An. strodei in the An. strodei subgroup and identifies four species under An. arthuri informally named A-D herein. The use of a multi-locus barcode is proposed for species identification, which has potentially important utility for vector incrimination. Individuals previously found naturally infected with Plasmodium vivax in the southern Amazon basin and reported as An. strodei are likely to have been from An. arthuri C identified in this study.

Phylogeny of Western Palaearctic long-eared bats (Mammalia, Chiroptera, Plecotus) - a molecular perspective

Die Phylogenie der Westpaläarktischen Langohren (Mammalia, Chiroptera, Plecotus) – eine molekulare Analyse Die Langohren stellen eine Fledermausgattung dar, die fast alle westpaläarktischen Habitate bist zum Polarkreis hin besiedeln und in vielerlei Hinsicht rätselhaft sind. In der Vergangenheit wurden zahlreiche Formen und Varietäten beschrieben. Trotzdem galt für lange Zeit, dass nur zwei Arten in Europa anerkannt wurden. Weitere Arten waren aus Nordafrika, den Kanaren und Asien bekannt, aber auch deren Artstatus wurde vielfach in Frage gestellt. In der vorliegenden Dissertation habe ich mittels molekularer Daten,der partiellen Sequenzierung mitochondrialer Gene (16S rRNA und ND1), sowie der mitochondrialen Kontrollregion, eine molekular Analyse der phylogenetischen Verwandtschaftsverhältnisse innerhalb und zwischen den Linien der westpaläarktischen Langohren durchgeführt. Die besten Substitutionsmodelle wurden berechnet und phylogenetische Bäume mit Hilfe vier verschiedener Methoden konstruiert: dem neighbor joining Verfahren (NJ), dem maximum likelihood Verfahren (ML), dem maximum parsimony Verfahren (MP) und dem Bayesian Verfahren. Sechs Linien der Langohren sind genetisch auf einem Artniveau differenziert: Plecotus auritus, P. austriacus, P. balensis, P. christii, P. sardus, und P. macrobullaris. Im Falle der Arten P. teneriffae, P. kolombatovici und P. begognae ist die alleinige Interpretation der genetischen Daten einzelner mitochondrialer Gene für eine Festlegung des taxonomischen Ranges nicht ausreichend. Ich beschreibe in dieser Dissertation drei neue Taxa: Plecotus sardus, P. kolombatovici gaisleri (=Plecotus teneriffae gaisleri, Benda et al. 2004) and P. macrobullaris alpinus [=Plecotus alpinus, Kiefer & Veith 2002). Morphologische Kennzeichen, insbesondere für die Erkennung im Feld, werden hier dargestellt. Drei der sieben Arten sind polytypisch: P. auritus (eine west- und ein osteuropäische Linie, eine sardische Linie und eine aktuell entdeckte kaukasische Linie, Plecotus kolombatovici (P. k. kolombatovici, P. k. gaisleri und P. k. ssp.) und P. macrobullaris (P. m. macrobullaris und P. m. alpinus). Die Verbreitungsgebiete der meisten Arten werden in dieser Arbeit erstmals ausschließlich anhand genetisch zugeordneter Tiere dargestellt.Die Untersuchung der ökologischen Einnischung der nun anerkannten Formen, insbesondere in Gebieten sympatrischer Verbreitung, bietet ein spannendes und lohnendes Feld für zukünftige Forschungen. Nicht zuletzt muss sich die Entdeckung eines beachtlichen Anteils kryptischer Diversität innerhalb der westpaläarktischen Langohren auch bei der Entwicklung spezieller Artenschutzkonzepte widerspiegeln.

Genetic diversity among horse populations with a special focus on the Franches-Montagnes breed

Genetic characterization helps to assure breed integrity and to assign individuals to defined populations. The objective of this study was to characterize genetic diversity in six horse breeds and to analyse the population structure of the Franches-Montagnes breed, especially with regard to the degree of introgression with Warmblood. A total of 402 alleles from 50 microsatellite loci were used. The average number of alleles per locus was significantly lower in Thoroughbreds and Arabians. Average heterozygosities between breeds ranged from 0.61 to 0.72. The overall average of the coefficient of gene differentiation because of breed differences was 0.100, with a range of 0.036-0.263. No significant correlation was found between this parameter and the number of alleles per locus. An increase in the number of homozygous loci with increasing inbreeding could not be shown for the Franches-Montagnes horses. The proportion of shared alleles, combined with the neighbour-joining method, defined clusters for Icelandic Horse, Comtois, Arabians and Franches-Montagnes. A more disparate clustering could be seen for European Warmbloods and Thoroughbreds, presumably from frequent grading-up of Warmbloods with Thoroughbreds. Grading-up effects were also observed when Bayesian and Monte Carlo resampling approaches were used for individual assignment to a given population. Individual breed assignments to defined reference populations will be very difficult when introgression has occurred. The Bayesian approach within the Franches-Montagnes breed differentiated individuals with varied proportions of Warmblood.

Phylogenetic Relationships of Clawed Lobster Genera (Decapoda : Nephropidae) Based on Mitochondrial 16S rRNA Gene Sequences

Approximately 350 base pairs (bp) of the mitochondrial 16S rRNA gene were used to study the phylogenetic relationships among 5 genera of the clawed lobster family Nephropidae (infraorder Astacidea), including Homarus, Homarinus, Metanephrops, Nephrops, and Nephropsis. Maximum-parsimony analysis, using a hermit crab, Pagurus pollicaris (infraorder Anomura), as an outgroup. produced a tree topology in which Homarus and Nephrops formed a well-supported clade that excluded Homarinus. The same tree topology was obtained from both neighbor-joining and maximum-likelihood analyses, Some morphological characters that appear synapomorphic for Nephrops and Metanephrops may be due to convergence rather than symplesiomorphy. The current taxonomy, therefore, does not reflect the phylogeny of this group as suggested by the molecular data. More molecular data and studies using homologous morphological characters me needed to reach a better understanding of the phylogenetic history of clawed lobsters.

Phylogenetic analysis of “Volvocacae” for comparative genetic studies

Sequence analysis based on multiple isolates representing essentially all genera and species of the classic family Volvocaeae has clarified their phylogenetic relationships. Cloned internal transcribed spacer sequences (ITS-1 and ITS-2, flanking the 5.8S gene of the nuclear ribosomal gene cistrons) were aligned, guided by ITS transcript secondary structural features, and subjected to parsimony and neighbor joining distance analysis. Results confirm the notion of a single common ancestor, and Chlamydomonas reinharditii alone among all sequenced green unicells is most similar. Interbreeding isolates were nearest neighbors on the evolutionary tree in all cases. Some taxa, at whatever level, prove to be clades by sequence comparisons, but others provide striking exceptions. The morphological species Pandorina morum, known to be widespread and diverse in mating pairs, was found to encompass all of the isolates of the four species of Volvulina. Platydorina appears to have originated early and not to fall within the genus Eudorina, with which it can sometimes be confused by morphology. The four species of Pleodorina appear variously associated with Eudorina examples. Although the species of Volvox are each clades, the genus Volvox is not. The conclusions confirm and extend prior, more limited, studies on nuclear SSU and LSU rDNA genes and plastid-encoded rbcL and atpB. The phylogenetic tree suggests which classical taxonomic characters are most misleading and provides a framework for molecular studies of the cell cycle-related and other alterations that have engendered diversity in both vegetative and sexual colony patterns in this classical family.

Did homeodomain proteins duplicate before the origin of angiosperms, fungi, and metazoa?

Homeodomain proteins are transcription factors that play a critical role in early development in eukaryotes. These proteins previously have been classified into numerous subgroups whose phylogenetic relationships are unclear. Our phylogenetic analysis of representative eukaryotic sequences suggests that there are two major groups of homeodomain proteins, each containing sequences from angiosperms, metazoa, and fungi. This result, based on parsimony and neighbor-joining analyses of primary amino acid sequences, was supported by two additional features of the proteins. The two protein groups are distinguished by an insertion/deletion in the homeodomain, between helices I and II. In addition, an amphipathic alpha-helical secondary structure in the region N terminal of the homeodomain is shared by angiosperm and metazoan sequences in one group. These results support the hypothesis that there was at least one duplication of homeobox genes before the origin of angiosperms, fungi, and metazoa. This duplication, in turn, suggests that these proteins had diverse functions early in the evolution of eukaryotes. The shared secondary structure in angiosperm and metazoan sequences points to an ancient conserved functional domain.

Evolutionary relationships of the coelacanth, lungfishes, and tetrapods based on the 28S ribosomal RNA gene.

The origin of land vertebrates was one of the major transitions in the history of vertebrates. Yet, despite many studies that are based on either morphology or molecules, the phylogenetic relationships among tetrapods and the other two living groups of lobe-finned fishes, the coelacanth and the lungfishes, are still unresolved and debated. Knowledge of the relationships among these lineages, which originated back in the Devonian, has profound implications for the reconstruction of the evolutionary scenario of the conquest of land. We collected the largest molecular data set on this issue so far, about 3,500 base pairs from seven species of the large 28S nuclear ribosomal gene. All phylogenetic analyses (maximum parsimony, neighbor-joining, and maximum likelihood) point toward the hypothesis that lungfishes and coelacanths form a monophyletic group and are equally closely related to land vertebrates. This evolutionary hypothesis complicates the identification of morphological or physiological preadaptations that might have permitted the common ancestor of tetrapods to colonize land. This is because the reconstruction of its ancestral conditions would be hindered by the difficulty to separate uniquely derived characters from shared derived characters in the coelacanth/lungfish and tetrapod lineages. This molecular phylogeny aids in the reconstruction of morphological evolutionary steps by providing a framework; however, only paleontological evidence can determine the sequence of morphological acquisitions that allowed lobe-finned fishes to colonize land.

The Allelic Abundance of Afghanistan, a Pool for West and South Eurasian Genes and a Blockade against Eastern Admixture

In this study, I divided samples from individuals within Afghanistan based upon geography (i.e., north versus south). I determined allelic frequencies and other statistical parameters for 15 STR loci (i.e., D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, Dl3S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, and FGA). I conducted pairwise comparisons with 19 neighboring Eurasian populations to assign Gstatistics and p-values. Categorizing the populations into five groups (i.e., Central Asia, East Asia, South Asia, the Middle East, and the Caucasus/Anatolia), I derived values for intra-population, inter-population, and total variance. Admixture analyses determined the highest allelic contributions to be from the Caucasus/ Anatolia, while negligible contributions were made by Central Asia and East Asia. A Correspondence Analysis revealed clustering of both northern and southern Afghanistan with Georgia, Turkey, northern Iran, and southern Iran of the Caucasus/ Anatolia and the Middle East. A Neighbor-Joining phylogenetic tree was constructed to generate bootstrap values over 1, 000 reiterations.

Filogenia molecular das enzimas isocitrato liase e malato sintase e sua evolução em Viridiplanta

The plant metabolism consists of a complex network of physical and chemical events resulting in photosynthesis, respiration, synthesis and degradation of organic compounds. This is only possible due to the different kinds of responses to many environmental variations that a plant could be subject through evolution, leading also to conquering new surroundings. The glyoxylate cycle is a metabolic pathway found in glyoxysomes plant, which has unique role in the seedling establishment. Considered as a variation of the citric acid cycle, it uses an acetyl coenzyme A molecule, derived from lipids beta-oxidation to synthesize compounds which are used in carbohydrate synthesis. The Malate synthase (MLS) and Isocitrate lyase (ICL) enzyme of this cycle are unique and essential in regulating the biosynthesis of carbohydrates. Because of the absence of decarboxylation steps as rate-limiting steps, detailed studies of molecular phylogeny and evolution of these proteins enables the elucidation of the effects of this route presence in the evolutionary processes involved in their distribution across the genome from different plant species. Therefore, the aim of this study was to establish a relationship between the molecular evolution of the characteristics of enzymes from the glyoxylate cycle (isocitrate lyase and malate synthase) and their molecular phylogeny, among green plants (Viridiplantae). For this, amino acid and nucleotide sequences were used, from online repositories as UniProt and Genbank. Sequences were aligned and then subjected to an analysis of the best-fit substitution models. The phylogeny was rebuilt by distance methods (neighbor-joining) and discrete methods (maximum likelihood, maximum parsimony and Bayesian analysis). The identification of structural patterns in the evolution of the enzymes was made through homology modeling and structure prediction from protein sequences. Based on comparative analyzes of in silico models and from the results of phylogenetic inferences, both enzymes show significant structure conservation and their topologies in agreement with two processes of selection and specialization of the genes. Thus, confirming the relevance of new studies to elucidate the plant metabolism from an evolutionary perspective

Variabilidade genética de Leishmania spp. circulantes entre seres humanos e cães e infecção de flebotomíneos em áreas endêmicas para as leishmanioses no Rio Grande do Norte

Leishmania infantum is the main etiologic agent of visceral leishmaniasis in the New World. The pattern of distribution of leishmaniasis has changed substantially and has presented an emerging profile within the periphery of the Large Urban Centers. Leishmania infection can compromise skin, mucosa and viscera. Only 10% of the individuals infected develop the disease and 90% of human infection is asymptomatic. The main factors involved in the development of the disease are the host immune response, the vector’s species and the parasite’s genetic content. The sequencing of Leishmania isolated seeks to increase the understanding of the symptoms of individuals. The aim of this study was to evaluate the genetic diversity of circulating Leishmania strains among humans, and symptomatic and asymptomatic, and dogs from endemic areas of Rio Grande do Norte State and analyze sandflies from endemic areas for cutaneous and visceral disease. The genetic variability was evaluated by the use of markers hsp70 , ITS1 and a whole genome sequencing was also carried out. The amplified hsp70 and ITS1 of samples were analyzed and assembled using a Phred / Phrap package. The dendograms were constructed using the same methodology, but adding 500 bootstraps, followed by inferences on the relationships between Leishmania variants. The sequences of the 20 Brazilian isolates were mapped to the reference genome L. infantum JPCM5, using the Bowtie2 program and the identification of 36 contigs. The information of the valid SNPs were used in the PCA. SNPs were visualized by Geneious 7.1 and IGV. The genome annotations were transferred to their respective chromosomes and displayed on Geneious. The matching sequences of all chromosomes were aligned using Mauve. The phylogenetic trees were calculated according to maximum likelihood and JTT models. Sandflies were analyzed by PCR for the identification of Leishmania infection, a blood meal source and GAPDH sand fly. As a result, hsp70 and ITS1 were not capable of identifying genetic variability among human isolates from symptomatic and asymptomatic, and dogs. The complete sequencing of the 20 Brazilian isolates revealed a strong similarity between the circulating Leishmania strains in Rio Grande do Norte. The isolates collected in the city of Natal from humans and canines remained grouped in all analyzes, suggesting that there is genotypic and geographic proximity among the isolates. The isolated samples in the 1990s had a higher genotypic diversity when compared to freshly isolated samples. All isolates presented 36 chromosomes with variable ploidy among them, no correlation was found between the number of amastina genes copies, gp63, A2 and SSG with such clinic forms. In general, we did not find correlation between symptomatic and asymptomatic clinical forms and the gene content of the Brazilian isolates of Leishmania. 34,28% of the sandflies collected in the upper west region were L. longipalpis and the main sources of blood meal were humans, dogs and chickens.

Variabilidade genética de Leishmania spp. circulantes entre seres humanos e cães e infecção de flebotomíneos em áreas endêmicas para as leishmanioses no Rio Grande do Norte

Leishmania infantum is the main etiologic agent of visceral leishmaniasis in the New World. The pattern of distribution of leishmaniasis has changed substantially and has presented an emerging profile within the periphery of the Large Urban Centers. Leishmania infection can compromise skin, mucosa and viscera. Only 10% of the individuals infected develop the disease and 90% of human infection is asymptomatic. The main factors involved in the development of the disease are the host immune response, the vector’s species and the parasite’s genetic content. The sequencing of Leishmania isolated seeks to increase the understanding of the symptoms of individuals. The aim of this study was to evaluate the genetic diversity of circulating Leishmania strains among humans, and symptomatic and asymptomatic, and dogs from endemic areas of Rio Grande do Norte State and analyze sandflies from endemic areas for cutaneous and visceral disease. The genetic variability was evaluated by the use of markers hsp70 , ITS1 and a whole genome sequencing was also carried out. The amplified hsp70 and ITS1 of samples were analyzed and assembled using a Phred / Phrap package. The dendograms were constructed using the same methodology, but adding 500 bootstraps, followed by inferences on the relationships between Leishmania variants. The sequences of the 20 Brazilian isolates were mapped to the reference genome L. infantum JPCM5, using the Bowtie2 program and the identification of 36 contigs. The information of the valid SNPs were used in the PCA. SNPs were visualized by Geneious 7.1 and IGV. The genome annotations were transferred to their respective chromosomes and displayed on Geneious. The matching sequences of all chromosomes were aligned using Mauve. The phylogenetic trees were calculated according to maximum likelihood and JTT models. Sandflies were analyzed by PCR for the identification of Leishmania infection, a blood meal source and GAPDH sand fly. As a result, hsp70 and ITS1 were not capable of identifying genetic variability among human isolates from symptomatic and asymptomatic, and dogs. The complete sequencing of the 20 Brazilian isolates revealed a strong similarity between the circulating Leishmania strains in Rio Grande do Norte. The isolates collected in the city of Natal from humans and canines remained grouped in all analyzes, suggesting that there is genotypic and geographic proximity among the isolates. The isolated samples in the 1990s had a higher genotypic diversity when compared to freshly isolated samples. All isolates presented 36 chromosomes with variable ploidy among them, no correlation was found between the number of amastina genes copies, gp63, A2 and SSG with such clinic forms. In general, we did not find correlation between symptomatic and asymptomatic clinical forms and the gene content of the Brazilian isolates of Leishmania. 34,28% of the sandflies collected in the upper west region were L. longipalpis and the main sources of blood meal were humans, dogs and chickens.

Genetic Diversity and Conservation of Pseudophoenix (Arecaceae) in Hispaniola

The Caribbean genus Pseudophoenix (Arecaceae) has its center of taxonomic diversity in Hispaniola (Haiti and the Dominican Republic). Three species (P. ekmanii, P. lediniana, and P. vinifera) are restricted to this island. In this thesis I investigated the population genetic diversity and structure of Pseudophoenix using ten microsatellite loci. Results showed homozygote excess and high inbreeding coefficients in all populations across all polymorphic loci. Overall, there was high differentiation among populations. Results from the Bayesian and Neighbor Joining cluster analyses identified groups that were consistence with currently accepted species delimitation. We included the only known population of an undescribed morph from the Dominican Republic that has been suggested to represent a new species. Results from the cluster analyses suggested that this putative species is closely related to P. sargentii from Turk and Caicos. Our study provided insights pertinent to the conservation genetics and management of this genus in Hispaniola.

SSR genetic diversity assessment of popular pigeonpea varieties in Malawi reveals unique fingerprints

Background: Pigeonpea ( Cajanus cajan L. Millsp.) is a drought tolerant legume of the Fabaceae family and the only cultivated species in the genus Cajanus. It is mainly cultivated in the semi-arid tropics of Asia and Oceania, Africa and America. In Malawi, it is grown as a source of food and income and for soil improvement in intercropping systems. However, varietal contamination due to natural outcrossing causes significant quality reduction and yield losses. In this study, 48 polymorphic SSR markers were used to assess the diversity among all pigeonpea varieties cultivated in Malawi to determine if a genetic fingerprint could be identified to distinguish the popular varieties. Results: A total of 212 alleles were observed with an average of 5.58 alleles per marker and a maximum of 14 alleles produced by CCttc019 (Marker 40). Polymorphic information content (PIC), ranged from 0.03 to 0.89 with an average of 0.30. A neighbor-joining tree produced 4 clusters. The most commonly cultivated varieties, which include released varieties and cultivated land races, were well-spread across all the clusters observed, indicating that they generally represented the genetic diversity available in Malawi, although substantial variation was evident that can still be exploited through further breeding. Conclusion: Screening of the allelic data associated with the five most popular cultivated varieties, revealed 6 markers – CCB1, CCB7, Ccac035, CCttc003, Ccac026 and CCttc019 – which displayed unique allelic profiles for each of the five varieties. This genetic fingerprint can potentially be applied for seed certification to confirm the genetic purity of seeds that are delivered to Malawi farmers.

Association of kinase insert domain-containing receptor (KDR) gene polymorphism/ haplotypes with recurrent spontaneous abortion and genetic structure

Background: Recurrent spontaneous abortion is one of the diseases that can lead to physical, psychological, and, economical problems for both individuals and society. Recently a few numbers of genetic polymorphisms in kinase insert domain-containing receptor (KDR) gene are examined that can endanger the life of the fetus in pregnant women. Objective: The risk of KDR gene polymorphisms was investigated in Iranian women with idiopathic recurrent spontaneous abortion (RSA). Materials and Methods: A case controlled study was performed. One hundred idiopathic recurrent spontaneous abortion patients with at least two consecutive pregnancy losses before 20 weeks of gestational age with normal karyotypes were included in the study. Also, 100 healthy women with at least one natural pregnancy were studied as control group. Two functional SNPs located in KDR gene; rs1870377 (Q472H), and rs2305948 (V297I) as well as one tag SNP in the intron region (rs6838752) were genotyped by using PCR based restriction fragment length polymorphism (PCR-RFLP) technique. Haplotype frequency was determined for these three SNPs’ genotypes. Analysis of genetic STRUCTURE and K means clustering were performed to study genetic variation. Results: Functional SNP (rs1870377) was highly linked to tag SNP (rs6838752) (D´ value=0. 214; χ2 = 16.44, p<0. 001). K means clustering showed that k = 8 as the best fit for the optimal number of genetic subgroups in our studied materials. This result was in agreement with Neighbor Joining cluster analysis. Conclusion: In our study, the allele and genotype frequencies were not associated with RSA between patient and control individuals. Inconsistent results in different populations with different allele frequencies among RSA patients and controls may be due to ethnic variation and used sample size.