This study aims to determine species-level relationships within the genus Heteromys, as well as generic-level relationships among members of the subfamily Heteromyinae using a phylogenetic framework. Molecular sequence data were generated from two mitochondrial genes (cytochrome b and cytochrome oxidase I) and three nuclear gene segments (β-fibrinogen, engrailed protein II, and myosin heavy chain II), and analyzed under maximum parsimony, maximum likelihood, and Bayesian optimality criteria to infer relationships. Chapter 1 focuses on the phylogenetic and taxonomic implications for Heteromys from the analyses of sequence data. Phylogenies also provided a framework for delimiting species boundaries within the wide-ranging Heteromys desmarestianus complex using the Wiens and Penkrot method. Several well-supported clades within this complex were recovered, including H. goldmani, H. nubicolens, and H. oresterus, as well as five groups identified as candidate species. Heteromys oasicus was not found to be genetically diagnosable from H. anomalus, and was relegated to subspecific status. I present a revised taxonomy as follows: the monotypic subgenus Xylomys is maintained (H. nelsoni); the subgenus Heteromys is divided into three species groups – anomalus (H. anomalus [including H. oasicus], H. australis, and H. teleus), desmarestianus (H. desmarestianus, H. goldmani, H. nubicolens, H. oresterus, and the five candidate species), and gaumeri (H. gaumeri). Chapter 2 describes phylogenetic inferences made from analyses of heteromyine taxa, genera Heteromys and Liomys. Many studies have recovered Liomys as paraphyletic relative to Heteromys, and the goal of this chapter was to address this taxonomic problem. The Liomys pictus species group (L. irroratus, L. pictus, and L. spectabilis) was recovered as sister to Heteromys rather than to the L. salvini group (L. adspersus and L. salvini). I recommend a revised taxonomy for the subfamily as follows: the genus Heteromys is retained as delineated in Chapter 1; the genus Liomys is reduced in scope to include only L. irroratus, L. pictus, and L. spectabilis; the subgeneric name Schaeferia is elevated to generic rank and includes S. adspersus and S. salvini. This classification better reflects the phyletic diversity within the subfamily Heteromyinae, and requires fewer name changes; thus providing nomenclatural stability.

A videira é uma das plantas mais antigas cultivadas pela humanidade, sendo que no Brasil a uva é a terceira fruta com maior volume de produção, atrás apenas do cultivo das bananas e das laranjas. Apesar da produção rentável, principalmente aos pequenos produtores, o parreiral é susceptível a várias doenças cujo manejo compromete até 59% dos gastos do produtor. No estado de São Paulo, dentre as doenças, três têm destaque: a antracnose (causada pelo Sphaceloma ampelinum), o míldio da videira (causado pelo Plasmopara viticola) e a ferrugem (causada pelo Phakopsora euvitis). Os produtores utilizam controle químico de forma intensa e preventiva, chegando a 100 aplicações de fungicidas em um ciclo de até 120 dias. Os principais fungicidas utilizados são os inibidores da quinona oxidase (QoI), que agem impedindo o transporte de elétrons do citocromo b ao citocromo c1 na cadeia respiratória da mitocôndria. Porém, existem relatos de resistência ao fungicida aplicado no campo em diversos países. As substituições G143A, G137R e F129L na sequência da proteína citocromo b impedem que o fungicida se ligue ao seu sítio alvo. As mutações que levam às substituições estão localizadas em uma das regiões chamada hotspot do gene citocromo b (cytB). Visto que, pela carência de estudos, a resistência genética a esses fungicidas nunca foi relatada no Brasil, o objetivo principal desse trabalho foi sequenciar e caracterizar a região hotspot em isolados de míldio, ferrugem e antracnose provenientes de parreirais do estado de São Paulo. Foram selecionados 35 isolados de 11 locais diferentes; desses, 11 isolados de míldio foram considerados geneticamente resistentes, pois apresentam a mutação para o resíduo alanina na posição 143, e 4 isolados foram considerados geneticamente sensíveis. Os dois isolados de ferrugem selecionados também foram considerados geneticamente sensíveis. Pela estratégia de Genome Walking foi possível sequenciar 65% do gene cytB de um dos isolados brasileiros de P. viticola; foram encontrados poucos polimorfismos e nenhum íntron na sequência analisada. Os resultados obtidos com esse estudo podem servir de suporte para a tomada de decisões de manejo mais adequadas para a realidade da viticultura brasileira; além disso, são importantes para futuros estudos sobre a evolução do patógeno com a pressão seletiva exercida pelos fungicidas. / Grapevine is one of the most ancient cultivated plants and its fruit, grape, is notably important in Brazil, since it is the third most produced, only behind banana and citrus. Although it is rentable especially to smallholders, the vineyard is often attacked by several pathogens and the damages induced by them can compromise up to 59% of the producers\' expenses in order to keep the diseases under control. In Sao Paulo state there are three important diseases that attack vineyards: anthracnose (caused by Sphaceloma ampelinum), downy mildew (caused by Plasmopara viticola) and rust (caused by Phakopsora euvitis). Pest management practices used by the producers relies on intensive and preventive use of fungicides, in which the culture is sprayed 100 times per vineyard\'s growth cycle (that last approximately 120 days). One of the most used fungicides are the quinone oxidase inhibitors (QoI), that act by blocking the electron transport chain at the mitochondria binding at the Qo site of the cytochrome b (cytB) complex. However, there are several reports of the presence of resistant strains in different countries. Resistance is caused by the aminoacids substitutions F129L, G137R and G143A in the cytochrome b protein sequence, that prevent the fungicide molecule binding to its target site. The mutations in the cytB gene that lead to these substitutions are harbored in a region called hotspot for fungicide resistance. Since this type of study was never reported in Brazil, the main purpose of this work was to sequence and characterize the hotspot region of different isolates from anthracnose, downy mildew and rust. Thirty five isolates from eleven different locations were choosen for the study. Eleven of them harbored the mutation that lead to the substitution G143A; these were then considered genetically resistant to the QoI fungicides. On the contrary, four downy mildew and the two rust isolates were considered sensitive to the QoI fungicides, since none of the aminoacids substitutions were observed. Also, by using a technique named Genome Walking it was possible to sequence 65% of cytB gene from a Brazilian downy mildew isolate. In this sequence were found few polymorphisms and none intron. These study findings are unique for Brazilian isolates and might be useful to provide reliable support for the pest management decisions regarding the reality that is found at the vineyards in Brazil. Furthermore, the results presented here are important to the comprehension of pathogen\'s evolution when suffering from a selective pressure caused by the intensive use of fungicides.

A videira é uma das plantas mais antigas cultivadas pela humanidade, sendo que no Brasil a uva é a terceira fruta com maior volume de produção, atrás apenas do cultivo das bananas e das laranjas. Apesar da produção rentável, principalmente aos pequenos produtores, o parreiral é susceptível a várias doenças cujo manejo compromete até 59% dos gastos do produtor. No estado de São Paulo, dentre as doenças, três têm destaque: a antracnose (causada pelo Sphaceloma ampelinum), o míldio da videira (causado pelo Plasmopara viticola) e a ferrugem (causada pelo Phakopsora euvitis). Os produtores utilizam controle químico de forma intensa e preventiva, chegando a 100 aplicações de fungicidas em um ciclo de até 120 dias. Os principais fungicidas utilizados são os inibidores da quinona oxidase (QoI), que agem impedindo o transporte de elétrons do citocromo b ao citocromo c1 na cadeia respiratória da mitocôndria. Porém, existem relatos de resistência ao fungicida aplicado no campo em diversos países. As substituições G143A, G137R e F129L na sequência da proteína citocromo b impedem que o fungicida se ligue ao seu sítio alvo. As mutações que levam às substituições estão localizadas em uma das regiões chamada hotspot do gene citocromo b (cytB). Visto que, pela carência de estudos, a resistência genética a esses fungicidas nunca foi relatada no Brasil, o objetivo principal desse trabalho foi sequenciar e caracterizar a região hotspot em isolados de míldio, ferrugem e antracnose provenientes de parreirais do estado de São Paulo. Foram selecionados 35 isolados de 11 locais diferentes; desses, 11 isolados de míldio foram considerados geneticamente resistentes, pois apresentam a mutação para o resíduo alanina na posição 143, e 4 isolados foram considerados geneticamente sensíveis. Os dois isolados de ferrugem selecionados também foram considerados geneticamente sensíveis. Pela estratégia de Genome Walking foi possível sequenciar 65% do gene cytB de um dos isolados brasileiros de P. viticola; foram encontrados poucos polimorfismos e nenhum íntron na sequência analisada. Os resultados obtidos com esse estudo podem servir de suporte para a tomada de decisões de manejo mais adequadas para a realidade da viticultura brasileira; além disso, são importantes para futuros estudos sobre a evolução do patógeno com a pressão seletiva exercida pelos fungicidas. / Grapevine is one of the most ancient cultivated plants and its fruit, grape, is notably important in Brazil, since it is the third most produced, only behind banana and citrus. Although it is rentable especially to smallholders, the vineyard is often attacked by several pathogens and the damages induced by them can compromise up to 59% of the producers\' expenses in order to keep the diseases under control. In Sao Paulo state there are three important diseases that attack vineyards: anthracnose (caused by Sphaceloma ampelinum), downy mildew (caused by Plasmopara viticola) and rust (caused by Phakopsora euvitis). Pest management practices used by the producers relies on intensive and preventive use of fungicides, in which the culture is sprayed 100 times per vineyard\'s growth cycle (that last approximately 120 days). One of the most used fungicides are the quinone oxidase inhibitors (QoI), that act by blocking the electron transport chain at the mitochondria binding at the Qo site of the cytochrome b (cytB) complex. However, there are several reports of the presence of resistant strains in different countries. Resistance is caused by the aminoacids substitutions F129L, G137R and G143A in the cytochrome b protein sequence, that prevent the fungicide molecule binding to its target site. The mutations in the cytB gene that lead to these substitutions are harbored in a region called hotspot for fungicide resistance. Since this type of study was never reported in Brazil, the main purpose of this work was to sequence and characterize the hotspot region of different isolates from anthracnose, downy mildew and rust. Thirty five isolates from eleven different locations were choosen for the study. Eleven of them harbored the mutation that lead to the substitution G143A; these were then considered genetically resistant to the QoI fungicides. On the contrary, four downy mildew and the two rust isolates were considered sensitive to the QoI fungicides, since none of the aminoacids substitutions were observed. Also, by using a technique named Genome Walking it was possible to sequence 65% of cytB gene from a Brazilian downy mildew isolate. In this sequence were found few polymorphisms and none intron. These study findings are unique for Brazilian isolates and might be useful to provide reliable support for the pest management decisions regarding the reality that is found at the vineyards in Brazil. Furthermore, the results presented here are important to the comprehension of pathogen\'s evolution when suffering from a selective pressure caused by the intensive use of fungicides.

Os insetos da subfamília Triatominae Jeannel, 1919 de Reduviidae (Hemiptera: Heteroptera: Reduviidae), também conhecidos como barbeiros, constituem um grupo amplamente distribuído pela região Neotropical que comporta hoje, 142 espécies ocorrentes e uma fóssil, distribuídas em 18 gêneros, são insetos hematófagos estritos em todas as fases da vida e reconhecidos vetores da Doença de Chagas. Essa enfermidade é considerada um dos mais importantes problemas de saúde na América Latina, com cerca de 12 a 14 milhões de indivíduos chagásicos, 60 milhões vivendo em risco e cerca de 20.000 caso/ano em 18 países da América do Sul e Central. Neste estudo foram analisadas seqüências de nucleotídeos dos genes 16S e CitB mitocondrial em populações de triatomíneos, dos gêneros Panstrongylus Berg, 1879, Rhodnius Stål, 1859 e Triatoma Laporte, 1832, mantidos em colônias no insetário Serviço Especial de Saúde de Araraquara (SESA) - SP, comparando-as com seqüências dos mesmos genes disponíveis no GenBank. Os fragmentos obtidos variaram de 311 a 317 pb (16S) e 393 pb (CitB). Observou-se baixa variação intra-específica entre as distâncias genéticas do gene 16S, enquanto o gene CitB mostrou-se mais polimórfico, podendo ser utilizado para estudos de populações geográficas. As seqüências geradas foram alinhadas com seqüências dos mesmos genes para outros triatomíneos e também de Arilus cristatus Linaeus, 1763 (Hemiptera: Reduviidae: Harpactorinae) (16S e CitB) e Oncerotrachelus sp. Stål, 1868 (Hemiptera: Reduviidae: Saicinae) (16S). As relações entre as espécies foram avaliadas pelos métodos de Distância (Neighbor Joining), Máxima Parcimônia e Máxima Verossimilhança. As análises de fragmentos para os dois genes demonstraram a parafilia de Rhodniini e Triatomini, confirmando resultados anteriores. Evidenciou-se a possível origem dos "Triatomas" Neotropicais a partir de derivações de Triatomas Neárticos, verificada pela estreita relação genética de Panstrongylus e Triatoma vitticeps (Stål, 1859) com o ramo Norte-Centro Americano de Triatoma; por meio das relações com T. protracta (Uhler, 1894), Dipetalogaster maxima (Uhler, 1894) e T. dimidiata Latreille, 1811. É dada a conhecer a primeira seqüência de bases nitrogenadas de fragmentos do gene 16S para Triatoma sherlocki Papa, Jurberg, Carcavallo, Cerqueira e Barata, 2002. Os resultados mostraram a utilidade de 16S e CitB como marcadores moleculares de espécies e de populações de triatomíneos e sua importância em questões de sistemática e taxonomia, no entanto, exigem um alto grau de segurança e controle de contaminação. Há necessidade de novos estudos envolvendo outros marcadores e o uso de caracteres sistemáticos clássicos de morfologia, ecologia e comportamento, indispensáveis para decisões sistemáticas adequadas uma vez, que teriam impacto não apenas sistemático mas, para as estratégias de controle. As colônias de triatomíneos do SESA revelaram-se importante fonte de material para estudos sistemáticos de triatomíneos, pois compreendem amostras significativas de várias populações triatomínicas da América, principalmente de espécies consideradas vetores principais do mal de Chagas como Triatoma infestans (Klug, 1834), Panstrongylus megistus (Burmeister, 1835), Rhodnius prolixus Stål, 1859 e Triatoma brasiliensis Neiva, 1911. Algumas questões sistemáticas relativas a origem e relacionamento filogenético dos triatomíneos continuam em aberto; novos reagrupamentos serão necessários para que a Subfamília continue válida; Análises mais completas, com a inclusão de representantes das tribos Alberproseniini, Bolboderini e Cavernicolini, são ainda necessárias. / The insects of the Triatominae subfamily Jeannel, 1919 of Reduviidae (Hemiptera: Heteroptera), also known as triatomine bugs, constitute a widely distributed group in the Neotropical region and including 142 current species and a fossil one, distributed in 18 genera. They are strict hematophagic insects in all phases of their life cicle and recognized vectors of the Chagas disease. This disease is considered one of the most serious Health problem in Latin America, with about 12 the 14 million of chagasic individuals, 60 million people living at risk and about 20,000 cases/year in 18 Central and South American countries. In this study mitocondrial 16S and CytB nucleotide sequences were analyzed in populations of triatomine bugs of the genera Panstrongylus Berg, 1879, Rhodnius Stål, 1859 and Triatoma Laporte, 1832 from colonies maintained in the Insectary of Special Health Service of Araraquara - SESA (acronym in Portuguese), Araraquara- SP, comparing them with sequences of the same available genes deposited in the GenBank. The obtained fragments varied from 311 to 317 bp (16S gene) and 393 pb (CytB). Low intra-specific variation among the genetic distance of the 16S gene, while the CytB gene revealed a higher polymorphism, being able to be used in geographic population studies. The generated sequences were aligned with sequences of the same genes for other triatomine bugs and also with Arilus cristatus Linaeus, 1763 (16S and CytB) and Oncerotrachelus sp Stål, 1868 (16S). The relationships among the species had been evaluated by the methods of Distance (Neighbor Joining), Maximum Parsimony and Maximum Likelihood. The analyses of fragments for the two genes had demonstrated the paraphily of Rhodniini and Triatomini, confirming previous results. It was hypothesized the possible origin of the Neotropical "Triatomas" from derivations of Nearctic triatomes, verified for the narrow genetic relation of Panstrongylus and Triatoma vitticeps (Stål, 1859) with the North-Center American branch of Triatoma by the relationships with T. protracta (Uhler, 1894), D. maxima (Uhler, 1894) and T. dimidiata Latreille, 1811. It is known the first sequence of nitrogen bases of fragments of the 16S gene for Triatoma sherlocki Papa, Jurberg, Carcavallo, Cerqueira e Barata, 2002. The results had shown the utility of 16S and CytB as markers of species and of triatomine populations and their importance in systematic and taxonomy questions. However, demands one high degree of security and control of contamination. There is a necessity of new studies involving other molecular markers and the use of classic systematic characters of morphology, ecology and behavior, indispensable for systematic decisions adjusted a time, that would have not only systematic impact but, for the control strategies. The colonies of triatomine of the SESA had shown important source of material for systematic studies of this kind of insect, since they include significant samples of triatomine populations of America, mainly of species considered the main vectors of the Chagas disease such as Triatoma infestans (Klug, 1834), Panstrongylus megistus (Burmeister, 1835), Rhodnius prolixus Stål, 1859 and Triatoma brasiliensis Neiva, 1911. Some systematic questions related to the origin and phylogenetic relationship of the triatomine bugs have kept opened; new regroupings will be necessary so that the Subfamily continues valid; more complete analyses, with the inclusion of representative members of the Alberproseniini, Bolboderini and Cavernicolini tribes, are still necessary.

This thesis was focused on suitability of selected molecular markers for detection of genetic variability of four species from the family Calliphoridae important in the forensic entomology. A set of eight markers, both mitochondrial (COI, ND6, CytB, CR) and nuclear (RP S12, RP S13, RP L12, PB2), were applied on samples from natural populations of representative species (Calliphora vicina, Lucilia caesar, Lucilia sericata, Phormia regina) most common in the Czech Republic. Level of detected variability was evaluated and compared, both with respect to the particular species as well as their geographic origin.

Os insetos da subfamília Triatominae Jeannel, 1919 de Reduviidae (Hemiptera: Heteroptera: Reduviidae), também conhecidos como barbeiros, constituem um grupo amplamente distribuído pela região Neotropical que comporta hoje, 142 espécies ocorrentes e uma fóssil, distribuídas em 18 gêneros, são insetos hematófagos estritos em todas as fases da vida e reconhecidos vetores da Doença de Chagas. Essa enfermidade é considerada um dos mais importantes problemas de saúde na América Latina, com cerca de 12 a 14 milhões de indivíduos chagásicos, 60 milhões vivendo em risco e cerca de 20.000 caso/ano em 18 países da América do Sul e Central. Neste estudo foram analisadas seqüências de nucleotídeos dos genes 16S e CitB mitocondrial em populações de triatomíneos, dos gêneros Panstrongylus Berg, 1879, Rhodnius Stål, 1859 e Triatoma Laporte, 1832, mantidos em colônias no insetário Serviço Especial de Saúde de Araraquara (SESA) - SP, comparando-as com seqüências dos mesmos genes disponíveis no GenBank. Os fragmentos obtidos variaram de 311 a 317 pb (16S) e 393 pb (CitB). Observou-se baixa variação intra-específica entre as distâncias genéticas do gene 16S, enquanto o gene CitB mostrou-se mais polimórfico, podendo ser utilizado para estudos de populações geográficas. As seqüências geradas foram alinhadas com seqüências dos mesmos genes para outros triatomíneos e também de Arilus cristatus Linaeus, 1763 (Hemiptera: Reduviidae: Harpactorinae) (16S e CitB) e Oncerotrachelus sp. Stål, 1868 (Hemiptera: Reduviidae: Saicinae) (16S). As relações entre as espécies foram avaliadas pelos métodos de Distância (Neighbor Joining), Máxima Parcimônia e Máxima Verossimilhança. As análises de fragmentos para os dois genes demonstraram a parafilia de Rhodniini e Triatomini, confirmando resultados anteriores. Evidenciou-se a possível origem dos "Triatomas" Neotropicais a partir de derivações de Triatomas Neárticos, verificada pela estreita relação genética de Panstrongylus e Triatoma vitticeps (Stål, 1859) com o ramo Norte-Centro Americano de Triatoma; por meio das relações com T. protracta (Uhler, 1894), Dipetalogaster maxima (Uhler, 1894) e T. dimidiata Latreille, 1811. É dada a conhecer a primeira seqüência de bases nitrogenadas de fragmentos do gene 16S para Triatoma sherlocki Papa, Jurberg, Carcavallo, Cerqueira e Barata, 2002. Os resultados mostraram a utilidade de 16S e CitB como marcadores moleculares de espécies e de populações de triatomíneos e sua importância em questões de sistemática e taxonomia, no entanto, exigem um alto grau de segurança e controle de contaminação. Há necessidade de novos estudos envolvendo outros marcadores e o uso de caracteres sistemáticos clássicos de morfologia, ecologia e comportamento, indispensáveis para decisões sistemáticas adequadas uma vez, que teriam impacto não apenas sistemático mas, para as estratégias de controle. As colônias de triatomíneos do SESA revelaram-se importante fonte de material para estudos sistemáticos de triatomíneos, pois compreendem amostras significativas de várias populações triatomínicas da América, principalmente de espécies consideradas vetores principais do mal de Chagas como Triatoma infestans (Klug, 1834), Panstrongylus megistus (Burmeister, 1835), Rhodnius prolixus Stål, 1859 e Triatoma brasiliensis Neiva, 1911. Algumas questões sistemáticas relativas a origem e relacionamento filogenético dos triatomíneos continuam em aberto; novos reagrupamentos serão necessários para que a Subfamília continue válida; Análises mais completas, com a inclusão de representantes das tribos Alberproseniini, Bolboderini e Cavernicolini, são ainda necessárias. / The insects of the Triatominae subfamily Jeannel, 1919 of Reduviidae (Hemiptera: Heteroptera), also known as triatomine bugs, constitute a widely distributed group in the Neotropical region and including 142 current species and a fossil one, distributed in 18 genera. They are strict hematophagic insects in all phases of their life cicle and recognized vectors of the Chagas disease. This disease is considered one of the most serious Health problem in Latin America, with about 12 the 14 million of chagasic individuals, 60 million people living at risk and about 20,000 cases/year in 18 Central and South American countries. In this study mitocondrial 16S and CytB nucleotide sequences were analyzed in populations of triatomine bugs of the genera Panstrongylus Berg, 1879, Rhodnius Stål, 1859 and Triatoma Laporte, 1832 from colonies maintained in the Insectary of Special Health Service of Araraquara - SESA (acronym in Portuguese), Araraquara- SP, comparing them with sequences of the same available genes deposited in the GenBank. The obtained fragments varied from 311 to 317 bp (16S gene) and 393 pb (CytB). Low intra-specific variation among the genetic distance of the 16S gene, while the CytB gene revealed a higher polymorphism, being able to be used in geographic population studies. The generated sequences were aligned with sequences of the same genes for other triatomine bugs and also with Arilus cristatus Linaeus, 1763 (16S and CytB) and Oncerotrachelus sp Stål, 1868 (16S). The relationships among the species had been evaluated by the methods of Distance (Neighbor Joining), Maximum Parsimony and Maximum Likelihood. The analyses of fragments for the two genes had demonstrated the paraphily of Rhodniini and Triatomini, confirming previous results. It was hypothesized the possible origin of the Neotropical "Triatomas" from derivations of Nearctic triatomes, verified for the narrow genetic relation of Panstrongylus and Triatoma vitticeps (Stål, 1859) with the North-Center American branch of Triatoma by the relationships with T. protracta (Uhler, 1894), D. maxima (Uhler, 1894) and T. dimidiata Latreille, 1811. It is known the first sequence of nitrogen bases of fragments of the 16S gene for Triatoma sherlocki Papa, Jurberg, Carcavallo, Cerqueira e Barata, 2002. The results had shown the utility of 16S and CytB as markers of species and of triatomine populations and their importance in systematic and taxonomy questions. However, demands one high degree of security and control of contamination. There is a necessity of new studies involving other molecular markers and the use of classic systematic characters of morphology, ecology and behavior, indispensable for systematic decisions adjusted a time, that would have not only systematic impact but, for the control strategies. The colonies of triatomine of the SESA had shown important source of material for systematic studies of this kind of insect, since they include significant samples of triatomine populations of America, mainly of species considered the main vectors of the Chagas disease such as Triatoma infestans (Klug, 1834), Panstrongylus megistus (Burmeister, 1835), Rhodnius prolixus Stål, 1859 and Triatoma brasiliensis Neiva, 1911. Some systematic questions related to the origin and phylogenetic relationship of the triatomine bugs have kept opened; new regroupings will be necessary so that the Subfamily continues valid; more complete analyses, with the inclusion of representative members of the Alberproseniini, Bolboderini and Cavernicolini tribes, are still necessary.

Today, DNA-barcoding with the gene COI is regularly used in the identification of fish. However, this is not an adequate way of identifying species of tuna due to COI lacking sufficient interspecies divergence. This is problematic since fraud and mislabeling are a major concern within the fish and tuna industries. Thus, there is a need for a new genetic barcode region when identifying the 15 tuna species within the tribe Thunnini. This study has considered six mitochondrial genetic regions (16S, ATP8, COII, CR, CytB, and ND2) and their potential as barcodes in comparison to COI. To be of practical use, the barcode has to be able to differentiate between all 15 tuna species, as well as contain conserved primer binding sites and be approximately 400 bp, or shorter. Analyses of the regions were made through Multiple Sequence Alignments built using ClustalW in Mega 11.0. The candidates were first evaluated through neighbor-joining trees and plots of inter- and intraspecies variation, and then analyzed further in search of conserved regions for primer binding, flanking a segment of approximately 400 bp (or shorter). This resulted in two possible barcode candidates with corresponding primers from the CR and ND2 genes. As a final step, these two were analyzed for specificity using BLAST, to evaluate their actual utility in differentiating the tuna species. The results show that they both can identify the different tuna species, but that ND2 is superior with 100% identification accuracy. In addition to the theoretical analysis, the ability of the primers was measured through a real PCR amplification. Unfortunately, only the CR barcode could be evaluated, but the results show it to be practically useful. Even though the utility of ND2 in PCR could not be analyzed, it is highly recommended as a region for further investigations. Given the strong theoretical support, it definitely shows promise as a new barcode for species identification of tuna.