Int.J.Curr.Microbiol.App.Sci (2014) 3(10) 156-168

ISSN: 2319-7706 Volume 3 Number 10 (2014) pp. 156-168 http://www.ijcmas.com

Review Article Approaches on ´s Molecular Systematics Studies

José Brites-Neto1,3, Gino Chaves da Rocha2, Luiz Humberto Gomes3 and Keila Maria Roncato Duarte4

1Epidemiological Surveillance Department, Secretary of Health, Americana, São Paulo, 2Faculty of Agronomy and Veterinary, University of Brasília, Brazil 3Department of Exact Sciences/ESALQ/USP, São Paulo, Brazil; 4Institute of Science of Nova Odessa, São Paulo, Brazil *Corresponding author

A B S T R A C T

Phylogenies moleculars provide means for analyzing many old problems of behavioral and eco-epidemiology of , many of which are importance for public health. A number of molecular markers is used in studies of phylogeny, K e y w o r d s ecology and population dynamics. Mitochondrial DNA is the most widely used Ribosomal marker of DNA regions for as well as for in general. Nuclear DNA, ribosomal RNA genes have been widely used, especially due to their abundance in mitochondrial the genome and its ease of amplification and sequencing of highly conserved and DNA, variable regions (containing the 18S, 5.8S and 28S genes separated by non polymorphis transcribed and transcribed spacers). Questions about the intraspecific population m, molecular structure, species limits, and the diagnosis of species, can be often elucidated by markers, techniques such as allozyme electrophoresis, restriction fragment length phylogenies. polymorphism (RFLP), or any variety of new techniques for DNA-based, as single strand conformation polymorphism (SSCP), amplified fragment length polymorphism DNA (AFLP) or random amplified polymorphic DNA (RAPD). Systematic studies on arthropods have analyzed about forty protein-coding genes, all major ribosomal RNA genes (mitochondrial and nuclear) as well as numerous non-coding regions. The regions most commonly sequenced on systematic in the arthropods are mitochondrial DNA and nuclear ribosomal DNA and have supported largely hypotheses based on morphology.

Introduction

Arthropods in general, and the fruit fly, of studies on vertebrates, as an important Drosophila melanogaster (Figure. 1A), in model of genetic organism. All the particular, have traditionally served as fundamentals of classical genetics were built models in developmental biology for largely through studies on these insects, understanding morphology or for biomedical where molecular genetic testing with this reasons providing a key to the development Diptera became the basis for understanding

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Int.J.Curr.Microbiol.App.Sci (2014) 3(10) 156-168 how all animals develop into adults from a some genes of mitochondrial DNA) are fertilized egg (Alberts, 2008). useful for comparison of the relationship between taxa and genes that evolve more Specific changes in the hereditary slowly are useful for analysis of distant taxa information expressed in families of mutant (Martins, 2007). flies, were correlated exactly in how the loss or modification of specific bands of their Phylogenies moleculars provide means for giant chromosomes (Figure. 1B) were analyzing many old problems of behavioral processed. Due to DNA replication without and eco-epidemiology of insects, many of intervention of a cell division, each which are importance for public health. The chromosome in these cells contained more principle of co-evolution was also examined than 1,000 aligned identical DNA from many perspectives. The phylogenetic molecules. The genes were transcribed at a research has showed that the association high rate corresponding to bands with a between organisms in many cases persisted bloated appearance. The bands stained dark for long periods of diversification. By brown in micrograph appeared as sites examining the relationships between social where a regulatory protein was bound to insects and their relatives, the molecular DNA. Once all Drosophila genes were trees casts a light on the origins of sociality sequenced (genome with 13,000 genes in different groups, as well as the evolution presenting 180 megabases), more than in of diversity of castes within certain social any other organism was possible to trace the groups (Caterino et al., 2000). chain of cause and effect of genetic instructions encoded in chromosomal DNA. Review of Studies Historically, studies by Thomas Hunt Morgan and colleagues in experiments with Molecular data have revolutionized our Drosophila melanogaster have proven the understanding of arthropod relationships chromosomal theory of inheritance with the since when systematists constructed hypothesis that the genes were located on arthropod phylogenies using nuclear chromosomes and were arranged linearly ribosomal genes, nuclear protein-encoding along them (Gilbert, 2003). genes, or a combination of these with mitochondrial genes. Developments in The position of arthropods among animals sequencing technology and shotgun has changed radically in the past two approaches following the sequencing of the decades as a result of refinements in first complete eukaryotic genomes changed cladistic analysis and especially by the our views on how to produce DNA sequence introduction of molecular systematic studies data (Giribet and Edgecombe, 2012). on insects, through the advent of polymerase chain reaction (PCR), the development of Caterino et al., (2000) analyzed 567 studies automated sequencing and the increasing of molecular systematic in insects checking computing power into a new science, large divergence in genes and regions bioinformatics (Giribet and Edgecombe, already sequenced, indicating a need of an 2004). approach more coordinated and standardized for development of phylogenetic hypotheses Basically, any selection of gene region in insects. They suggested the use of studied in molecular analysis becomes cytochrome oxidase I gene, 16S, 18S and essential, since genes of rapid evolution (as the elongation factor 1-alpha, as patterns of

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Int.J.Curr.Microbiol.App.Sci (2014) 3(10) 156-168 molecular systematics complementary to the 12S and A + T) and Coleoptera (COI, COII, morphological and ecological studies, on COIII, ND5, ND4, Cyt b, ND1, 16S, 12S substantial contributions to evolutionary and A + T). biological processes. Due to the recognition that trees based on A number of molecular markers is used in mitochondrial gene may represent only a studies of phylogeny, ecology and partial and biased view on the phylogeny of population dynamics, such as internal organisms, many nuclear genes have been transcribed spacer (ITS). The ribosomal analyzed for the phylogeny of arthropods. DNA (rDNA) is composed of long arrays of Nuclear ribosomal RNA genes have been tandem repeat units separated by a non- widely used, especially due to their transcribed intergenic spacer (IGS or NTS) abundance in the genome and its ease of which has a variable number of small amplification and sequencing of highly internal sub-repetitions, and that are conserved and variable regions (containing variables on length between related species the 18S, 5.8S and 28S genes separated by closely and individuals of the same species, non transcribed and transcribed spacers). and external transcribed spacer (ETS). Each transcribed unit contains the sequences that Revised data also showed all regions of encoding to three ribosomal genes 18S, 5.8S ribosomal DNA in Entognatha, Apterygota and 28S and which are highly conserved and and Paleoptera (18S and 28S), essentially invariables. The first internal Orthopteroidea (18S, ITS2 and 28S), transcribed spacer (ITS1) is located between Hemipteroidea (18S, ITS1, 5.8S and ITS2), genes 18S e 5.8 S and the second internal Coleoptera (18S, ITS1, 5.8S, ITS2 and 28S), transcribed spacer (ITS2) between genes Lepidoptera (18S, ITS1 and 28S), Diptera 5.8S e 28S. Both can be used to differentiate (18S, ITS1, 5.8S, ITS2 and 28S), strains and subspecies of arthropods, (18S, ITS1, 5.8S, ITS2 and including mosquitoes and . 28S), Neuropteroidea, Strepsiptera, Mecoptera, Trichoptera and Siphonaptera Mitochondrial DNA (mtDNA) is the most (18S, 5.8S and 28S). widely used marker of DNA regions for insects as well as for animals in general. Nuclear protein-encoding genes which This popularity stems largely from their exhibit a wide variety of evolutionary rates apparent ease of isolation and amplification, have been evaluated as phylogenetic tool. even from little preserved specimens. These loci showed a high possibility for Revised data showed all mitochondrial resolving relationships in very conserved regions used in Lepidoptera (COI, COII, levels, in comparison to mitochondrial ND5, Cyt b, ND1, 16S, 12S and A + T), proteins. The rate variation among codon Siphonaptera (COII) and Neuroptera (ND2 positions in genes encoding proteins and COII), Entognatha (COI, COII, Cyt b, suggests that many of these loci can be 16S and 12S), Paleoptera (COI, COII and effective in elucidating the most recent 16S), Orthopteroidea (COI, COII, Cyt b, divergences. 16S and 12S), Hemipteroidea (COI, COII, Cyt b, ND1, 16S and 12S), Hymenoptera Caterino et al., (2000) also reviewed studies (ND2, COI, COII, ATpase6, COIII, ND6, that used as molecular markers, several Cyt b, ND1 and 16S), Diptera (ND2, COI, nuclear gene regions that encoding proteins COII, COIII, ND5, ND4, Cyt b, ND1, 16S, (Table 1).

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Questions about the intraspecific population generated by rearrangements or mutations structure, species limits, and the diagnosis of (insertions or deletions) between the two species, can be often elucidated by locations or at the site of hybridization of the techniques such as allozyme electrophoresis, two primers. Differences in only one base restriction fragment length polymorphism pair (point mutations) may be sufficient to (RFLP), or any variety of new techniques inhibit amplification. The low for DNA-based, as single strand reproducibility of RAPD is a much debated conformation polymorphism (SSCP), question. Many insects of different orders amplified fragment length polymorphism were analyzed by RAPD with different DNA (AFLP) or random amplified approaches, among which include polymorphic DNA (RAPD). The best phylogenetic studies, taxonomic studies and strategy will often be to use one or more of epidemiological studies, to obtain genomic these techniques in combination with DNA fingerprints of individuals, for analysis of sequencing, which has become a preference structure and genetic diversity of technique for generating comparative populations, to construct genetic maps of molecular data, consolidating the unique high resolution and on studies related to the perspective to the conduction of acquisition of insecticide resistance. Among evolutionary processes diversification of the studied species can highlight the DNA. mosquito vector of malaria, Anopheles, the mosquito vector of dengue and yellow fever, RFLP Restriction Fragment Length Aedes and the transmitter triatomine of Polymorphism Chagas disease, Triatoma. Insects of economic interest, such as Haematobia With the advent of techniques for irritans were also studied. In this technique sequencing, the data generated with a single oligonucleotide (containing 10 to 20 restriction enzymes disappeared on bases) is randomly constructed, resulting in phylogenetic studies insects, though the the amplification of a target sequence technique is even commonly applied in unknown. Each primer used directs the parallel studies. With the mapping of amplification of several DNA segments restriction sites, however, they still provide a simultaneously in different parts of the quick alternative for studying sequence genome, resulting in several bands in the variation in large cross-sections of the gel. genome, and have proved very useful and reasonably accurate for phylogeny The polymorphism produced by RAPD reconstruction. The most common use of technique appears in the form of presence or restriction enzymes in systematic is for the absence of fragments or bands in the gel. diagnostic profiles that are sought within the Unlike other methods, RAPD does not DNA fragments (more often in mtDNA). require preliminary work, such as construction of genomic banks, since it does RAPD - Random Amplified Polymorphic not require the previous development of a DNA library of probes specific for the organism of interest, eliminating the need to transfer The polymorphism of RAPD markers is DNA to membranes (Southern Blot) and the revealed by amplification of chromosomal use of radioactive isotopes or non- loci using primers composed of short radioactive marking. Another feature of this sequences of oligonucleotides and are molecular tool is that these markers are

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Int.J.Curr.Microbiol.App.Sci (2014) 3(10) 156-168 inherited as dominant markers. This Perspectives inheritance model does not allow differentiating heterozygous and Currently, molecular biology techniques homozygous dominant individuals without have been widely used for the purpose of subsequent crossings. By observing a band assisting in the identification of insects of on the gel, it is not possible to distinguish forensic importance. The mitochondrial whether that segment was originated of one DNA (mtDNA) has proved an excellent or two copies of the amplified sequence. molecular marker, particularly due to the simple and uniform organization of the SSCP Single Strand Conformation mitochondrial genome, the low number of Polymorphism recombinations and the high rate of nucleotide substitutions. In addition, a wide These techniques rely on differences in the range of universal primers is available for DNA secondary structures due to the mtDNA of insects, thereby permitting primary sequence variation and the trusted amplifications of genes or consequent variation in the rate of homologous regions in several groups of electrophoretic mobility. SSCP may be insects. The ability to recover, efficiently, sensitive to the differences of a single base genetic information from damaged or poorly pair. In entomological applications SSCP is preserved samples also facilitates the use of typically used in conjunction with mtDNA markers in forensic investigations. amplification of a specific fragment for the The combination of molecular methods such purpose of diagnosing species. SSCP also as polymerase chain reaction and restriction provides a quick method of differentiation of fragment length polymorphism (PCR-RFLP) polymorphisms between populations and has been very useful for identifying of species that can be sequenced for cryptic species or at different stages of life phylogenetic effects. A particularly (Thyssen et al., 2005; Thyssen, 2008). interesting application employs SSCP as an alternative to cloning to isolation nuclear In studies in Diptera (Calliphoridae), two alleles. specific regions of the mitochondrial DNA, the cytochrome oxidase subunit I (COI) and AFLP - Amplified fragment length the control region (CR) were amplified by polymorphism PCR and digested using restriction endonucleases. The cleavage patterns AFLP is a technique developed that as generated by endonucleases DraI and SspI RAPD, quickly locates genetic were suitable to distinguish the two species polymorphisms dispersed among organisms. Hemilucilia (Thyssen et al., 2005). Despite the apparent promise for applications entomological, AFLP has not Low et al., (2014) presented in their studies been widely applied on studies of systematic a molecular evidence of that the closely of insects. The technique is based on the related species Haematobia irritans property of certain restriction enzymes to Linnaeus and Haematobia exigua de leave, after cleavage of the DNA, cohesive Meijere are genetically distinct and could be ends of the known sequence. Thus, it is differentiated based on multilocus sequence possible to construct nucleotide sequences analysis of five mitochondrial genes, of double-stranded that bind at the ends of mitochondria-encoded cytochrome c oxidase the restriction fragments. subunit I (COI), cytochrome B (CytB),

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NADH dehydrogenase subunit 5 (ND5); lineages were studied with extraction nuclear encoded 18S ribosomal RNA (18S) protocols and standardization of DNA and and 28S ribosomal RNA (28S) genes. PCR using 11 markers selected by greater Results of these analyses demonstrated that polymorphism and consistency of 26 ISSR ND5 and CytB genes were more variables markers, which produced 172 polymorphic and informatives in resolving the bands with a variable genetic similarity interspecific relationships of both species, between 19% to 96%, allowing us to the COI gene was more valuable in inferring conclude that the ISSR technique can the intraspecific relationships and the efficiently identify DNA polymorphism in ribosomal 18S sequences and 28S of the two Trichogramma, with demonstration of species were highly conserved with intra- results that suggest an accentuated variation and inter-specific variation limited. inter and intraspecific. According to Wei et al., (2014), in animal Mitochondrial DNA fragment of about 700 mitochondrial genomes, gene arrangements bp, comprising part of the cytochrome are usually conserved across major lineages oxidase I, tRNA of leucine-full and part of but might be rearranged within derived the cytochrome oxidase II, was amplified by groups and provide valuable phylogenetic PCR, demonstrating that the application of characters. The mitochondrial genomes of molecular tools has been extremely useful Cephalonomia gallicola (Chrysidoidea: for separating species and groups of species Bethylidae) and Wallacidia oculata of ants of the genus Linepithema (Martins, (Vespoidea: Mutillidae) were sequenced, 2007). where in Cephalonomia at least eleven tRNA and two protein-coding genes were Rampelotti et al., (2008) studied the genetic rearranged and in the Hymenoptera three diversity of Tibraca limbativentris events of rearrangement occured in the (Hemiptera: Pentatomidae) of Santa protein-coding genes (reversal, transposition Catarina and , using and reverse transposition). RAPD markers, aiming to test DNA extraction protocols and characterize Recent study in Anopheles cruzii (Diptera: populations of this important pest of Culicidae), the primary vector of malaria in rice. It wasn't observed relationship between humans and nonhuman primates in Brazil, geographical distance and the genetic through a multilocus analysis using six similarity of populations, reflecting a different fragments of nuclear genes was dispersion model of T. limbativentris still conducted to compare two populations unknown where studies exploring strategies (Itaparica and Florianópolis), representing of dispersal of the species could help in respectively species of the northeast and understanding the insect distribution, south. Were used, three fragments of genes evidencing which one the main source of involved in the control of circadian rhythms, genetic variability. timeless (tim), Clock (Clk) and cycle (cyc), and three genes encoding ribosomal Borba et al., (2005) analyzed the genetic proteins, Rp49 (Ribosomal protein 49, also dissimilarity of lineages by Trichogramma known as RpL32 - Ribosomal protein L32), (Hymenoptera: Trichogrammatidae) by RpS2 (Ribosomal protein S2) and RpS29 molecular marker ISSR, measuring the level (Ribosomal protein S29). This study was of genetic differentiation of these lineages crucial for analysis of differences between by employing the technique of ISSR. Five populations and helped to verify when the

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Int.J.Curr.Microbiol.App.Sci (2014) 3(10) 156-168 differentiation in the circadian genes is successful in correctly identifying greater than the divergence in the specimens. This construction of a COI constitution of loci, such as the genes identification system will also enable encoding ribosomal proteins Rp49, RpS29 important new insights into the and RpS2 (Rona et al., 2010). diversification of life and in the rules of molecular evolution (Hebert et al., 2003). Carapelli et al., (2006) demonstrated the existence of conflicts between The use of DNA barcode (Hebert et al., morphological and molecular data, as well 2003) has been widely discussed by those as between different data sets and methods who successfully apply this approach to of analysis, in respect to the basal phylogeny identify and diagnose species, and those of hexapods. However, molecular data have who believe that there are so many problems led to reconsideration of the hypothesis of in using this molecular marker that is not systematists of hexapods by longtime, based justified its use. For insects of the order on morphological data. This leads directly to Lepidoptera neither side seems completely the question whether it would be Hexapoda right or wrong, and although some groups a monophyletic taxon, or alternatively, one have been solved by taxonomically unique or more independent lineages within or additional use of this marker, for others Pancrustacea (Figure. 2), suggesting the the "barcode" helped little to solve development of more moleculars markers taxonomic problems. The "DNA barcode" is and the obtaining of a sampling of taxon a small genic region with 648 bp of the denser for both genes mitochondrial and mitochondrial DNA gene cytochrome c nuclear, well as a reassessment of new and oxidase 1 (COI) (Silva-Brandão, 2009). established morphological characters. Hiragi et al., (2009) analyzed the genetic On molecular studies in populations of variability of different populations of Aedes "horn fly" was cloned and sequenced the aegypti using RAPD markers (Random gene that confers one of types of resistance Amplified Polymorphic DNA), where DNA to pyrethroids ("knock down resistance to ten larvae collected from three different gene" kdr). Since then it is possible to populations were analyzed using ten RAPD detect this type of resistance (kdr) to primers, indicating the existence of genetic pyrethroids from a PCR, determining the variability inter and intrapopulation. frequency of kdr for susceptible and resistant populations (Sabatini, 2003). Oliveira et al., (2002) compared simple methods of preservation of specimens of Although much biological research to Dalbulus maidis (Hemiptera: Cicadellidae) depend on the diagnosis of the species, the regarding to quantity and quality of DNA taxonomic expertise is collapsing. The only for use in RAPD-PCR after periods prospect for a sustainable identification successive of storage. capability will be to build systems that employ DNA sequences as taxon 'barcodes'. Arias et al., (2003) emphasized the The mitochondrial gene cytochrome c importance of the mitochondrial genome oxidase I (COI) may serve as the nucleus of and the different analysis applied to it, to a global system for bio-identification solve issues of population and evolutionary animal. A COI profile model, based on the character in stingless bees addressing a analysis of a single individual from each of population analysis of remota, a the 200 species of Lepidoptera, was 100% phylogenetic analysis among species of

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Int.J.Curr.Microbiol.App.Sci (2014) 3(10) 156-168 genera Plebeia, and Phylogenetic relationships of 75 arthropod and the characterization of the mitochondrial and five species "outgroup" were analyzed genome of Melipona bicolor by sequencing, under likelihood criteria using four evidencing translocations of genes into strategies, where the aligned sequences for tRNA. Restriction maps for all of these 62 nuclear genes encoding proteins showed species were determined using 17 restriction results that strongly support the monophyly enzymes and the Southern blot technique to of Hexapoda; in contrast to mitochondrial identification of the fragments. For a better studies that place Collembola among positioning of restriction sites, was used a crustaceans, instead of other hexapods RFLP-PCR, which also allowed to locate (Regier et al., 2010). restrictions sites with relation to the genes present in the amplified fragment. Fernandez et al., (2005) analyzed the genomic polymorphism in Bombyx mori Ceretti-Junior et al., (2008) performing an through RAPD profile generated from the analysis of taxonomic and systematic genomic DNA of two lineages of Japanese relationships among species of triatomine caterpillars (which had a high content of the bugs (Hemiptera, Reduviidae) from colonies silk cocoon) and two lineages of Chinese of the Special Health Service Araraquara, caterpillars (who had greater resistance to demonstrated the utility of the 16S gene as a field cultivation); using the CSGE technique marker of species of triatomine and their (Conformation Sensitive Gel Eletrophoresis) importance in matters of systematic and which allows the identification of , emphasizing the need to be polymorphisms in target genes correlated further studies involving other markers with phenotype. Through this methodology associated with classic systematic characters was established the detection of co- of morphology, ecology and behavior for dominance, allowing the identification of appropriate systematic decisions, since it two alleles of the same locus and allowing would impact not systematic only but also the detection of polymorphic sequences of for control strategies. DNA (heteroduplex or homoduplex), with potential applications in breeding programs From the studies of Garcia and Powell of these lineages. Fet et al., (2003) discussed (1998) and Stothart et al., (1998) was the first phylogenetic hypotheses about inaugurated the era cladistic molecular on scorpions of the family Buthidae (17 genera) the systematic studies of Triatominae, presenting arguments based on molecular solving many impasses related to the status data (16S rRNA mitochondrial DNA). of some species or in respect to the cryptic species with intensification of the discussion Borges et al., (2010) developed a about the evolutive origin of this group and phylogenetic hypothesis based on support for their evidences of polyphyly or mitochondrial DNA from 21 species of paraphyly. New analyzes with the use of a Tityus collected in Venezuela, Trinidad, greater number of genetic markers Brazil and Panama, including 12 toxics taxa associated with the classic systematic to humans, through phylogenetic patterns of morphology, ecology and reconstruction based on 850 nucleotides of behavior, must be added for a suitable the cytochrome oxidase subunit I (COI) and phylogenetic decision, since haven't only 16S rRNA genes for the most of these important systematic, but serious species of scorpions. implications regarding to measures to control of vector species. 163

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Table.1 Nuclear protein-encoding genes used in molecular systematics of insects (Caterino et al., 2000)

Locus Sequences Taxa -amylase (amy) 38 Diptera, Coleoptera Acetylcholine esterase (achE) 21 Diptera Actin 5 Hymenoptera, Lepidoptera Alcohol dehydrogenase (adh) 157 Diptera Arylphorin (hexamerin family) 12 Lepidoptera Cecropin family 36 Diptera Chorion genes: s18, s15, s19 5 Diptera Cpnl-1 (anonymous nuclear DNA) 8 Orthoptera Dopa decarboxylase (ddc) 57 Lepidoptera Lepidoptera, Hymenoptera, Hemiptera, Elongation factor-1-alpha (EF-1 ) 196 Collembola, Archaeognatha, Blattodea EF-1 (copy 2) 1 Hymenoptera Esterase (est) 31 Diptera Glycerol-3-phosphate dehydrogenase (gpdh ou g3pdh) 39 Diptera Glycerol-6-phosphate dehydrogenase (g6pdh) 13 Diptera, Lepidoptera, Thysanura Guanylate cyclase 13 Diptera Globin family 33 Diptera Histone H1 8 Diptera Histone H4 7 Hymenoptera Hunchback (hb) 39 Diptera Kruppel 3 Diptera Luciferase 2 Coleoptera Lysozyme intron 16 Lepidoptera Myosin alkali light chain (Mic 1) intron 30 Diptera Nullo 7 Diptera Opsin 28 Hymenoptera, Diptera Period (per) 111 Diptera, Lepidoptera Phosphoglucose isomerase (Pgi) 2 Orthoptera Lepidoptera, Trichoptera, Diptera, Phosphoenolpyruvate carboxykinase (PEPCK) 12 Mecoptera, Siphonaptera Prune 10 Diptera Resistance to dieldrin (Rd1) 17 Coleoptera Retrotransposon reverse transcriptase 30 Hymenoptera Ribosomal protein 49 (rp49) 9 Diptera Serendiptiy- 7 Diptera Cu, Zn superoxide dismutase (sod) 30 Diptera Lepidoptera, Hemiptera, Blattodea, Sodium channel para locus1 27 Coleoptera Snail 3 Diptera Timeless 1 Diptera Triosephosphate isomerase (Tpi) 39 Diptera Vestigial (vg) 5 Diptera White 15 Diptera Wingless (wg) 57 Lepidoptera, Diptera Xanthine dehydrogenase (xdh) 37 Diptera Yolk protein 1 (yp1) 55 Diptera Yolk protein 2 (yp2) 40 Diptera Zeste 24 Diptera

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Figure.1 Giant chromosomes (B) in cells of the salivary gland of Drosophila melanogaster (A) (Alberts, 2008)

Figure.2 Different hypotheses of phylogenetic relationships in Hexapoda based on molecular data (Carapelli et al., 2006)

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According to Burger et al., (2012), our the partial mitochondrial genomes of a understanding of the phylogenetic further five species of soft ticks was used relationships among lineages of ticks has to test the controversial classification for been limited by the lack of resolution the genus-level in this group. The provided by phylogenetic markers most comparison of mitochondrial rRNA, commonly used. Mitochondrial genomes nuclear rRNA and the analyses of have been increasingly used to address the mitochondrial genome showed that only controversial phylogenetic relationships. mitochondrial genome sequences have the In this sense, presented a study on the potential to resolve the controversial nucleotide sequences of the complete phylogenetic relationships within the mitochondrial genomes of five species of major soft lineages (Burger et al., ticks metastriata: elaphense, 2014b). Amblyomma fimbriatum, Amblyomma sphenodonti, Bothriocroton concolor e According to Nava (2009) the molecular Bothriocroton undatum, aiming at markers most used in studies at population addressing the phylogenetic position of level and to infer inter and intra-specific two morphologically "primitive" species - variation of ticks were the ITS1 and ITS2 A. elaphense e A. sphenodonti - with and the mitochondrial genes 16S, 12S, regard to the genus Amblyomma. The cytochrome oxidase I (COI) and analysis of these five mitochondrial cytochrome oxidase III (COIII), and other genomes and more eleven other genetic markers such as the mitochondrial genomes of ticks, as well as microsatellites, allozyme polymorphism, the analysis of rRNA nuclear genes, random amplified polymorphic DNA generated strong evidence that genus (RAPD), polymorphism of the bm86 gene Amblyomma would be polyphyletic with and 28S rDNA gene. the inclusion of the species A. sphenodonti and A. elaphense. The diversity of available markers, no doubt, has favored the cause of the Burger et al., (2014a) using phylogenetic systematic, however, this pleiad of analyses of mitochondrial genome markers also carries on the risk of sequences, to approach the phylogenetic plurality, to the extent that the use of relationships among the species of the different estimators between the studies subgenus Boophilus, revealed a species difficults the comparison and makes it complex called Rhipicephalus microplus difficult their synthesis. The essence of complex (R. annulatus, R. australis and science is repeatability, and into their two clades of R. microplus), concluding interest we should reiterate some that cox1 and 16S rRNA were more principles integrated to the molecular successful in resolving the phylogenetic assays for phylogenetic studies. The relationships than the 12S rRNA or the objective should be always identify a ITS2 nuclear marker. relatively small set of markers to serve as a reference standard for possible Genetic analyses of soft ticks (Argasidae) comparisons. have been limited to 16S rRNA which is not highly informative, phylogenetically. References The sequence of entire mitochondrial genomes of seven species of soft ticks and Alberts, B. 2008. Molecular biology of the cell, 5th ed., Garland science, Taylor &

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