sdv.ba
MG R S De Mendonça 
  
Taxonômicos Estudos 
  
E De 
  
E Filogenia 
  
Genética Estrutura 
  
Ácaro Do 
  
Urticae Rajado 
  
INFERIDAS A PARTIR DE SEQUÊNCIAS DO DNA RIBOSSÔMICO E MITOCONDRIAL Koch 
  
Doutorado Tese 
  
T Kanzawai Kishida 
  
T Truncatus 
  
Ehara As 
  
Oligonychus Anonae Paschoal 
  
; O Mangiferus 
  
( Rahman 
  
Baker Sapra Tetranychus Bastosi Tuttle 
  
T Desertorum Banks 
  
T Evansi Baker 
  
T Ludeni Zacher 
  
T Mexicanus 
  
T Gloveri Baker 
  
M Tanajoa 
  
Planaltina 
  
Gilo Solanum 
  
Gilo Raddi 
  
Desertorum 
  
E Banksi 
  
T Desertorum + 
  
Recanto Das Emas 
  
Alpinia Purpurata 
  
K Schum 
  
Anona Squamosa 
  
O Anonae # 
  
O Anonae 
  
D Navia 
  
L C Miranda 
  
L Osbeck 
  
T Ludeni 
  
T Urticae 
  
  
  
  
  
  
  
  
  
  
  
  
  
BRAZIL WITH EMPHASIS IN THE PHYLOGENY AND POPULATION GENETIC STRUCTURE OF THE

Keywords: Acari, Tetranychinae, Bryobiinae, genetic variability, molecular markers, Neotropical region, misidentification. xi phytophagous mites, taxonomy, Tetranychini, Hystrichonychini, Eustachys distichophylla, Vitis vinifera Eurytetranychini, Hystrichonychini, Neotropical region, South America, taxonomy, Tetranychinae, Tetranychini

The data from the experiments was obtained by collecting mites from the Tetranychidae family in Brazil, in countries of the Palearctic region, and in Canada.

The intention was to obtain samples of the Tetranychus urticae Koch.

The main tasks undertaken whilst carrying out research for the thesis were: processing and inspecting samples from the Tetranychidae mites that were collected in different regions and in host plants; preserving Tetranychidae mites on microscope slides; morphological identification of the samples collected; perform molecular analyses (extraction, PCR amplification and sequencing of DNA) of specimens identified as T. urticae; conduct phylogenetic analysis of sequence data. In order to obtain comprehensive information on the genetic variability, phylogeny and structure of the population of T. urticae in Brazil and elsewhere, the sequences obtained for this study were analyzed together with sequences of closely related species of Tetranychus sensu stricto Tuttle and Baker (1968), available at Genbank, including those of T. cinnabarinus Boisduval, a supposed synonym. 1 Chapter 1.

 3. Phylogeny tree inferred from mtDNA COI sequences of Tetranychus urticae retrieved from Genbank and obtained in this study from T. urticae field samples. The colors highlight the haplotypes 1, 2 and 10.The haplotype number and Genbank accessions are given (see Table 6). The Neighbor-Joining (NJ) method was used based on distances calculated using Kimura's two-parameter (K2P) correction. Bootstrap values (higher than 50%) are near to the branches. The scale bar indicates the number of changes per site in NJ(K2P) inference ……………………. 

LIST OF TABLES -CHAPTER 4

Table 1. Collection records of Tetranychus urticae mites from Brazil sequenced in this study sequenced and respective information on amplified region……………………………………….….. Table 2. Collection records of Tetranychus urticae mites from Paleartic region and Canada sequenced and respective information on amplified region…………………….…………………….. Table 3. PCR and sequencing primers used to obtain nuclear ribosomal ITS and mitochondrial COI sequences of Tetranychus urticae………………………………………………………………….. Table 4. Descriptive analyses and molecular diversity indices. Intraspecific information based on ITS1 and ITS2 regions of rDNA for Tetranychus urticae sequences………………………………… Table 5. Descriptive analyses and molecular diversity indices. Intraspecific information based on sequences of the COI region of Tetranychus urticae specimens…………………………………….. Table 6. Accession numbers of COI sequences retrieved from Genbank for a set of Tetranychus urticae. The haplotype number attributed in this study and the respective country and host plant in Genbank are indicated. The sequences obtained in this study were included…………………... 

LIST -SUPPLEMENTAL MATERIAL -CHAPTER 4

Supplemental Material. Appendix 1. List of Tetranchus urticae ITS sequences retrieved from Genbank. Accession numbers were copied from Genbank. Information on host plants (common and family names), sample localities, the size of the sequence (bp) and original bibliographic references are given ……………………………………………………………………………………… Supplemental Material. Appendix 2. List of Tetranchus urticae COI sequences retrieved from Genbank. Accession numbers were copied from Genbank. Information on host plants (common and family names), sample localities, the size of the sequence (bp) and original bibliographic references are given …………………………………………………………………………………...…. vii xii localidade geográfica. O efeito da planta hospedeira não foi observado. A diversidade haplotípica, inferida pelas duas regiões do genoma (ITS e COI) analisadas foi maior entre os países banhados pelo mar Mediterrâneo. Não se observou variabilidade genética entre as populações de T. urticae coletadas nas cinco Regiões do Brasil quando se avaliou a região ITS. A presença de dois haplótipos mitocondriais (COI) foi constatada no Brasil, um compartilhado com a França, Espanha e Ilhas Canárias e outro com o Japão.

Palavras chaves: Acari, Bryobiinae, erros de identificação taxonômica, marcadores moleculares, região Neotropical, Tetranychinae, variabilidade genética. La famille des Tetranychidae est constituée d'environ 1250 espèces d'acariens phytophages. Parmi elles, une centaine est des ravageurs plus ou moins importants pour l'agriculture mondiale. L´acarien jaune, Tetranychus urticae Koch (Prostigmata: Tetranychidae) est une espèce ubiquiste qui est responsable de dommages importants qu´elle cause aux cultures. Divers aspects de la systématique, de la biologie et du contrôle des populations de cette espèce font actuellement l´objet de nombreuses recherches. Au Brésil, T. urticae fait partie des trois principaux acariens ravageurs des cultures. Malgré les avancées incontestables des études sur les tétranychidés, il est important d´étendre les recherches à de nouvelles régions du Brésil et pour des plantes hôtes n´ayant pas encore été évaluées. Les aspects moléculaires pour accedér des informations sur la relation phylogénétique entre d'espéces et sur la structure génétique des populations de ravageur, tel que T. urticae, peut également améliorer la compréhension de la famille. Dans cette étude, un inventaire des Tetranychidae du Brésil a été réalisé dans 15 États ainsi que dans le District Fédéral, avec 550 échantillons de 120 espèces végétales collectés. Des infestations par des acariens tétranychidés ont été confirmées dans 207 de ces échantillons. Vingt-deux espèces de tétranyques appartenant à sept genres de la sous-familles des Bryobiinae et Tetranychinae ont été identifiées chez 58 hôtes différents. Trente-six nouvelles plantes hôtes pour 11 espèces de tétranychidés ont été répertoriées au Brésil, en Amérique du sud ou dans le monde. De nouvelles localités ont été enregistrées au Brésil pour quatre tétranychides et une espèce a également été signalée pour la première fois en Amérique du sud. Quatre nouvelles espèces ont été découverte: deux appartenant au genre Oligonychus Berlese, présentes sur la vigne (Vitis vinifera L.) et le rosier dans le Minas Gerais; deux appartenant aux genres Monoceronychus McGregor et Schizotetranychus Tragardh, toutes deux présentes sur Eustachys distichophylla Lag. Nees, dans Rio Grande do Sul. La contribution à la systématique du genre Tetranychus a consisté à analyser conjointement des séquences d´ADN ribosomique (ITS) et mitochondrial (COI) de femelles de T.urticae, T. cinnabarinus (synonyme potentiel de T. urticae) et d'espèces très proches appartenant également au groupe Tetranychus sensu stricto. Les séquences on été obtenues à partir d'individus de T. urticae collectés dans cette étude et identifiés sans ambiguïté par des critères morphologiques et dans Genbank pour les autres espèces (105 ITS et 96 COI). Cette étude a mis en évidence des incohérences ce qui nous a amené à remettre en question la fiabilité des données moléculaires concernant le groupe Tetranychus s. str. disponibles dans Genbank. Nos résultats suggèrent que près de 30% des identifications taxonomiques parmi les séquences analysées sont fausses ou douteuses, surtout pour T. urticae, T. cinnabarinus, T. kanzawai Kishida et T. truncatus Ehara. Cette étude apporte également un éclairage nouveau sur le taxon controversé qu'est T. cinnabarinus et souligne la nécessité de l'utilisation combinée des approches morphologiques et moléculaires afin d'augmenter la fiabilité des séquences dans les banques de xiv données moléculaires publiques. Des données sur la variabilité génétique, la phylogénie et la structure de populations de T. urticae au Brésil et dans le monde sont ensuite présentées. De nouvelles séquences ITS et COI de 22 populations collectées au Brésil et de 37 prélevés dans la zone Paléarctique (France, Espagne, Iles Canaries, Grèce, Syrie, Tunisie, Pologne et Norvège -plus une du Canada) ont été analysés conjointement avec les séquences T. urticae déposées dans Genbank. Les résultats indiquent la présence significative d'une structuration génétique des populations de T. urticae par rapport à la localisation géographique. Cependant, l´effet de la plante hôte n´est pas observé. La diversité haplotypique, inférée à partir des ces deux régions du génome (ITS et COI) est plus élevée dans les pays du pourtour méditerranéen. La région ITS n´a pas permis d´observer de variabilité génétique entre les populations analysées. On a constaté la présence de deux haplotypes mitochondriaux (COI) au Brésil, l´un partagé avec la France, l´Espagne et les îles Canaries, et l´autre avec le Japon. Une discussion porte sur la nécessité de mener de nouvelles recherches en incluant d´autres régions du monde n´ayant pas encore été échantillonnées, ainsi que sur l'intérêt d´augmenter le nombre de populations dans certains pays déjà étudiés.

Mots-clés: Acari, Bryobiinae, Tetranychinae, erreurs d´identification, marqueurs moléculaires, région Néotropicale, variabilité génétique xv

Introduction et contexte de la thèse

Tetranychus urticae Koch 1836 est une espèce qui présente une grande importance agronomique au même titre que les autres de la famille Tetranychidae [START_REF] Helle | Spider mites: their biology, narural enemies and control[END_REF]. Cet acarien, qui est probablement originaire d´Eurasie, se serait disséminé par des moyens naturels dans les régions tempérées de l´hémisphère nord par le biais des activités humaines (Navajas et al. 1998). Cette espèce très polyphage a une distribuition cosmopolite (Bolland et al. 1998;Migeon & Dorkeld 2006). En conséquence il cause de sérieux dommages à de nombreuses cultures dans le monde entier et il est considéré comme l´un des acariens phytophages les plus importants [START_REF] Helle | Spider mites: their biology, narural enemies and control[END_REF], Moraes & Flechtmann 2008).

Au Brésil, en raison du nombre élevé d´espèces végétales à valeur économique qu'il attaque et en raison des dommages qu'il occasionne, T. urticae fait partie des trois principaux acariens ravageurs (Moraes & Flechtmann 2008).

De nombreux aspects portant sur la biologie et les habitudes alimentaires de T. urticae ont fait l´objet de recherches. Cette espèce suscite un intérêt tout particulier en ce qui concerne la sélection d'hôtes et les processus évolutifs puisque des différences sont rapportées entre des lignées (considérées comme des races) associées à des hôtes distincts [START_REF] Gotoh | Host race formation in Tetranychus urticae: genetic differentiation, host plant preference, and mate choice in a tomato and a cucumber strain[END_REF]. L´existence de différences génétiques parmi des populations en fonction de l'éloignement géographique, indépendamment de la plante hôte, est également mentionnée [START_REF] Tsagkarakou | Population structure in the spider mite Tetranychus urticae on Crete based on multiple allozymes[END_REF]. En plus de la différentiation génétique associée à la sélection par la plante hôte et par l'éloignement géographique, la présence de biotypes résistants à des pesticides est rapportée dans les pays où T. urticae a subi une intense pression de sélection par la lutte chimique [START_REF] Miller | Effect of early season immigration on cyhexatin and fometanate resistance of Tetranychus urticae (Acari: Tetranychidae) on strawberry in central California[END_REF][START_REF] Edge | Organo-tin resistance in two-spotted spider mite, Tetranychus urticae Koch (Acarina: Tetranychidae) in Australia[END_REF][START_REF] Dennehy | Laboratory and field investigations of spider mite (Acari: Tetranychidae) resistance to the selective acaricide propargite[END_REF][START_REF] Grafton-Cardwell | Spider mite species (Acari: Tetranychidae) response to propargite basis for an acaricide resistance management program[END_REF][START_REF] Flexner | Field reversion of organotin resistance in twospotted spider mite following relaxition of selection pressure[END_REF]Tian et al. 1992;[START_REF] Landeros | Susceptibility and mechanism of resistance of Tetranychus urticae (Tetranychidae) populations from greenhouse roses[END_REF][START_REF] Lee | Acaricide resistance of Tetranychus urticae in apple orchads in Korea: implications for an acaricide resistance management[END_REF]. Les problèmes de résistance de T. urticae aux acaricides au Brésil ont été étudiés par [START_REF] Sato | Resistência de Tetranychus urticae (Koch,1836) a acaricidas, em pessegueiro (Prunus Persica (L.) Batch) em Paranapanema e Jundiaí[END_REF][START_REF] Sato | Propargite resistance in Tetranychus urticae Koch (Acari: Tetranychidae) in the state of São Paulo, Brazil[END_REF][START_REF] Sato | Selections for fenpyroximate resitance and susceptibility, and inheritance, crossresistance and stability of fenpyroximate resistance in Tetranychus urticae Koch (Acari: Tetranychidae)[END_REF][START_REF] Sato | Seleções para resistência e suscetibilidade, detecção e monitoramento da resistência de Tetranychus urticae ao acaricida clorfenapir[END_REF] et par [START_REF] Flores | Estabilidade, seleção e padrão de herança da resistência do ácaro rajado, Tetranychus uricae Koch (Acari: Tetranychuidae), ao Abamectin[END_REF]. D'autre part, dans quelques ouvrages et publications, deux formes de l'espèce distinguées par la couleur du corps sont reconnues: la forme jaune correspondant à T. urticae et la forme rouge que certains acarologues considèrent comme une espèce à part entière appelée T. cinnabarinus (Boisduval 1867) (Boudreaux 1956;Boudreaux et Dosse 1963a, b, Meyer, 1974). Toutefois, les xvi différences morphologiques entre les deux formes étant très ténues et la coloration n'étant pas un critère taxonomique fiable, la validité du taxon T. cinnabarinus est rejetée par de nombreux auteurs (Pritchard et Baker 1955;Dupont 1979;Meyer, 1987;Baker & Tuttle 1994, Bolland et al., 1998). Actuellement, le problème de la synonymie entre ces deux espèces demeure un sujet de controverses.

Malgré l´importance économique de l´acarien jaune, il n´existe pas d´informations sur sa dissémination, sa sélection par les hôtes et sur la variabilité génétique dans différentes cultures et diverses régions géographiques au Brésil. Ces informations sont importantes car elles peuvent être à la base de l'élaboration de techniques spécifiques de contrôle de l´espèce notamment par le biais de la lutte intégrée contre les ravageurs. Elles sont également essentielles pour guider les mesures de mise en quarantaine visant à éviter l´introduction de nouveaux biotypes particulièrement agressifs, ou résistants à des pesticides.

Les populations de T. urticae et d'autres Tetranychidae sur les plantes cultivées sont devenues récurrentes que récemment notamment pour le soja cultivé dans le Rio Grande do Sul. Ces infestations semblent se produire plus fréquemment en fonction de l´avancée des zones de plantation mais aussi avec la mise en culture de nouvelles variétés ou en raison de modifications dans le système de conduite des cultures [START_REF] Guedes | Ácaros associados à cultrura da soja no Rio Grande do Sul[END_REF].

Parmi les 1265 espèces de Tetranychidae répertoriées dans le Monde près de 100 sont signalées au Brésil (Bolland et al. 1998;Migeon & Dorkeld 2008). Comme la majeure partie des connaissances sur les tétranychidés s'applique à la région du Sud-est brésilien ou a des plantes cultivées, il est important d´étendre les connaissances faunistiques sur les acariens de cette famille aux autres régions du pays.

Objectifs et approches d'étude

Ce projet de recherche s'est fixé plusieurs objectifs: 1) appréhender la diversité des acariens Tetranychidae et leur spectre d'hôtes au Brésil par des inventaires réalisés dans différentes régions; 2) contribuer à la systématique moléculaire du genre Tetranychus; 3) participer à l'évaluation de la variabilité xvii génétique des populations de T. urticae à l´échelle mondiale en y incluant des populations du Brésil et 4) rechercher les effets liés à la géographie et à la plante hôte dans la structure génétique des populations de T. urticae.

Deux marqueurs moléculaires indépendants ont été utilisés pour les études de variabilité, de caractérisation génétique et de structuration des populations de T. urticae: (1) un fragment de l´ADN mitochondrial, la région codant pour la Cytochrome oxydase I (COI); (2) la région du génome nucléaire "Internal Transcribed Spacer" (ITS) de l´ADN ribosomique (Navajas 1998;Navajas et al. 1998;2003) 1 Chapter 1. Renata S. Mendonça, Denise Navia, Carlos H. W. Flechtmann (2009). Two new spider mites (Acari: Tetranychidae) from Brazil -a Monoceronychus McGregor (Bryobinae) from fingergrass and an Oligonychus Berlese (Tetranychinae) from grape. International Journal of Acarology. 36 [START_REF]Among the pairs sampled, approximately 20% indicated a high level of distance (≥ 6%)[END_REF]. 2 Chapter 2. Renata S. Mendonça, Denise Navia, Ivone R. Diniz, Carlos H. W. Flechtmann (2010). Notes on Brazilian spider mites (Acari: Tetranychidae) -new hosts and localities. Journal of Insect Science. 3 Chapter 3. Renata S. de Mendonça, Denise Navia, Ivone R. Diniz, Philippe Auger, Maria Navajas (2010). A critical review of some closely related species of Tetranychus sensu stricto Tuttle & Baker (1968) (Acari: Tetranychidae) in the public DNA sequences databases.

. Experimental and Applied Acarology.

INTRODUCTION

The two-spotted spider mite Tetranychus urticae Koch, 1836, is a very important species to agriculture, as others species from the Tetranychidae family (Jeppson et al. 1975;[START_REF] Helle | Spider mites: their biology, narural enemies and control[END_REF]. This mite probably originated in Eurasia in light of the high frequency of samples from this region and from the variety of host plants on which it has been collected. It has been spread throughout the temperate regions of the northern hemisphere by wind and throughout the world via the transport of plants by man (Navajas 1998). Now it is a species of cosmopolitan distribution and highly polyphagous, infesting both cultivated and non-cultivated plants (Bolland et al. 1998;Migeon & Dorkeld 2006). For this reason and owing to the serious damage it causes to various agricultural crops throughout the world, it is regarded as one of the main phytophagous mites [START_REF] Helle | Spider mites: their biology, narural enemies and control[END_REF]. In Brazil, because of the high number of economically valuable vegetable species that are attacked and damaged, T. urticae is considered one of the three main pest mites in the country (Moraes & Flechtmann 2008) and its infestations has been recorded in many crops [START_REF] Flechtmann | Ácaros de importância agrícola[END_REF][START_REF] Sato | Gene flow amosng Tetranychus urticae (Acari: Tetranychidae) populations in Greece[END_REF][START_REF] Gallo | Manual de entomologia agrícola[END_REF].

Many aspects of the biology and feeding habits of T. urticae have been the focal point of research. Results regarding host selection and evolutionary processes have revealed differences between strains of T. urticae, which in some cases were regarded as races associated with distinct hosts [START_REF] Gotoh | Host race formation in Tetranychus urticae: genetic differentiation, host plant preference, and mate choice in a tomato and a cucumber strain[END_REF]). However research also mentioned genetic differences among populations due to the distance between the geographical regions analyzed, regardless of the host plant [START_REF] Tsagkarakou | Population structure in the spider mite Tetranychus urticae on Crete based on multiple allozymes[END_REF]. Besides genetic differences due to host plant selection and geographic regions, the presence of biotypes resistant to pesticides has been reported in countries where T. urticae experienced intense selection pressure [START_REF] Miller | Effect of early season immigration on cyhexatin and fometanate resistance of Tetranychus urticae (Acari: Tetranychidae) on strawberry in central California[END_REF][START_REF] Edge | Organo-tin resistance in two-spotted spider mite, Tetranychus urticae Koch (Acarina: Tetranychidae) in Australia[END_REF][START_REF] Dennehy | Laboratory and field investigations of spider mite (Acari: Tetranychidae) resistance to the selective acaricide propargite[END_REF][START_REF] Grafton-Cardwell | Spider mite species (Acari: Tetranychidae) response to propargite basis for an acaricide resistance management program[END_REF][START_REF] Flexner | Field reversion of organotin resistance in twospotted spider mite following relaxition of selection pressure[END_REF]Tian et al. 1992;[START_REF] Landeros | Susceptibility and mechanism of resistance of Tetranychus urticae (Tetranychidae) populations from greenhouse roses[END_REF][START_REF] Lee | Acaricide resistance of Tetranychus urticae in apple orchads in Korea: implications for an acaricide resistance management[END_REF]. Problems related to T. urticae resistance to insecticides/miticides in Brazil have been documented by [START_REF] Sato | Resistência de Tetranychus urticae (Koch,1836) a acaricidas, em pessegueiro (Prunus Persica (L.) Batch) em Paranapanema e Jundiaí[END_REF][START_REF] Sato | Propargite resistance in Tetranychus urticae Koch (Acari: Tetranychidae) in the state of São Paulo, Brazil[END_REF][START_REF] Sato | Selections for fenpyroximate resitance and susceptibility, and inheritance, crossresistance and stability of fenpyroximate resistance in Tetranychus urticae Koch (Acari: Tetranychidae)[END_REF][START_REF] Sato | Seleções para resistência e suscetibilidade, detecção e monitoramento da resistência de Tetranychus urticae ao acaricida clorfenapir[END_REF] and [START_REF] Flores | Estabilidade, seleção e padrão de herança da resistência do ácaro rajado, Tetranychus uricae Koch (Acari: Tetranychuidae), ao Abamectin[END_REF]. Despite some local differences among T. urticae populations, DNA sequence analysis of mites collected at a continental scale, tent to show high homogeneity of the species resulting from intense movements often human mediated (Navajse et al , 1988). Two forms of the species can be recognized by their green or red body colour. According to De [START_REF] De Boer | Reproductive barriers[END_REF], the body colours observed in mites result from the pigments found in the hemolymph. However, the red type of T. urticae has been often called T. cinnabarinus (Boisduval 1867) (Boudreaux 1956;Boudreaux & Dosse 1963a, b, Meyer 1974). The accuracy of the taxon T. cinnabarinus has been questioned by many authors (Pritchard and Baker 1955;Dupont 1979;Meyer 1987;Baker & Tuttle 1994) and it remains to this day a point of discussion.

Despite the economic importance of the two-spotted spider mite, there is no information regarding its genetic variability in different crops and geographic regions of Brazil. This kind of information is important and would be helpful in adopting phytosanitary measures designed to manage the mite. It is also essential in justifying quarantine methods to prevent introducing new T. urticae biotypes that are adapted to and aggressive towards different hosts, or resistant to different pesticides, as reported in other countries.

Infestations by the two-spotted spider mite and other Tetranychidae have posed economic problems in crops where damage was previously either not observed or only limited to specific areas, as in soybeans cultivated in Rio Grande do Sul. These infestations probably take place because of expanding cultivated areas, adapting new varieties, and/or changes crops management [START_REF] Guedes | Ácaros associados à cultrura da soja no Rio Grande do Sul[END_REF].

Approximately 100 described species of Tetranychidae have already been reported in Brazil (Bolland et al. 1998;Migeon & Dorkeld 2008). However, the most intensive search for Tetranychidae mites in the country has been concentrated mostly on the southeast region and usually pertains to economically important crops.

Agriculture has expanded towards new regions in Brazil and is encroaching biological reserves, it is important to continue studying tetranychids in unexplored areas and to gather new information that would enhance our understanding of this family.

GENERAL OBJECTIVES

The general objectives of this research were: cooperate to the understanding of mites from the Tetranychidae family in Brazil; contribute to the molecular systematics of the genus Tetranychus; obtain information on the genetic variability of T. urticae populations in a global context, including new populations from Brazil and the Palearctic region, and investigate the geographic and the host plant effects upon the genetic structure of T. urticae populations.

STRATEGIES

To conduct studies on Tetranychidae mites in Brazil, samples of mites were collected from several hosts ranging from ornamental, fruit-bearing, vegetable, grass, oilseed, and large crop species plants found in five regions in Brazil that included 15 states and the Federal District. With the support of the Embrapa Team of Genetic Resources and Biotechnology, and collaborators from universities and research institutes, a wide range of sampling was carried out in the country. Plants were collected when they presented symptoms of tetranychid attacks, according to Moraes & Flechtmann (2008), and the target organism was the two-spotted spider mite, T.

urticae.

For studies on variability, genetic characterization, and population structure of T. urticae, two target DNA fragments were PCR-amplified and sequenced: (1) the intergenic "Internal Transcribed Spacer" (ITS) of the nuclear ribosomal DNA; (2) a fragment of mitochondrial DNA, the Cytocrome Oxidase I (COI) gene; (Navajas 1998;Navajas et al. 1998;Navajas & Boursot 2003) This study reported new taxonomic information on tetranychid mites in Brazil.

It has uncovered that several DNA sequences that were publically available and attributed to economically relevant taxa, are incorrectly named. It also sheds new light on the controversial taxonomic status of T. cinnabarinus and highlights the need to use combined morphological and molecular approaches to ensure the reliability of access to public databases. The genetic variability, phylogeographic patterns, and the association to host plants in T. urticae populations collected in several regions of the world were investigated. 

THE CONTEXT OF THE THESIS

The Tetranychidae Donnadieu family includes a large group of phytophagous mites (spider mites) (Jeppson et al. 1975;[START_REF] Helle | Spider mites: their biology, narural enemies and control[END_REF]. Thus far, this family includes about 1,265 species, distributed in 76 genera. South America has recorded 186 tetranychid species, 105 of them being in Brazil (Bolland et al. 1998;Migeon & Dorkeld 2006).

Among the phytophagous mites of the Tetranychidae family, the genus Tetranychus Dufour 1832 includes 148 valid species (Migeon & Dorkeld 2006) and comprises several species of high economic and quarantine importance in agriculture. This genus provides a good example of organisms where species identification might be misleading. Systematics has traditionally relied on the diversity of a few minute morphological characters, mainly based on microscopic examination.

Not only males might be rare and difficult to find in field populations, but also the shape of the aedeagus (part of the male genitalia) must be known in order to discriminate closely related species. Thus, both sexes are often needed to confirm specie identification. As a result, morphologically based identification of Tetranychus species requires advanced taxonomic expertise, especially in morphologically similar taxa, as it is the case with species belonging to the group Tetranychus (Tetranychus) (Tuttle & Baker 1968), or Tetranychus sensu stricto, from the ninth species-group as defined by Flechtmann & Knihinicki (2002). In this group, the two-spotted spider mite, Tetranychus urticae, a highly polyphagous species causing outbreaks in many crops worldwide, is certainly the most studied species.

The wide range of geographical and ecological diversity of the two-spotted spider mite has prompted many studies on its morphological traits (Boudreaux 1956;[START_REF] Bund | Investigations on the Tetranychus urticae complex in North West Europe[END_REF]Boudreaux andDosse 1963a, b, Pritchard &Baker 1955;Meyer 1974Meyer , 1987;;Baker & Tuttle 1994;Ehara 1999;Carbonelle et al. 2007) and on its molecular systematics (Gotoh & Tokioka 1996;Navajas et al. 1996;Navajas et al. 1998, Ehara 1999;Hinomoto et al. 2001;[START_REF] Xie | Genetic relationship between the carmine spider mite Tetranychus cinnabarinus (Boisduval) and the two-spotted mite T. urticae Koch in China based on the mtDNA and rDNA ITS2 sequences[END_REF]Li et al. 2009).

Pest status of the two-spotted spider mite, Tetranychus urticae Koch 1836

Outbreaks and economical impact in worldwide agriculture

The two-spotted spider mite, Tetranychus urticae (Prostigmata: Tetranychidae) infests around 1,094 vegetable species in 109 countries around the world.. Among the 19 most important agricultural crops in world agriculture, six (citrus, cotton, grapes, apples, beans, and papaya) were reported as being hosts of the two-spotted spider mite (Yaninek & Moraes 1991). Damage caused by T. urticae is usually described in many other crops; however damage resulting by its infestations is not always quantified.

In cotton, T. urticae infestations have cause reductions of 14% to 44% in seed production, 4% in fat content, 17% in fiber weight (Cannerday & Arant 1964) as well as 4% in fiber resistance [START_REF] Oliveira | Ação dos ácaros Polyphagotarsonemus latus (Banks, 1904) e Tetranychus (T.) urticae Koch, 1836 na depreciação quantitativa e qualitativa da produção algodoeira. Tese Doutorado[END_REF], and 25% in weight loss [START_REF] Oliveira | Ação do ácaro "rajado" Tetranychus urticae (Koch, 1836) na depreciação quantitativa da produção algodoira[END_REF].

In ornamental plants, damages brought upon cloves, for example, reached between 20% and 30% [START_REF] Rajashekharappa | Estimation of loss caused due to spider mite, Tetranychus urticae koch (Acari: Tetranychidae) infestation on different varieties of carnation grown in polyhouse condition[END_REF].

Damage by T. urticae to citrus plants has been noted by [START_REF] Mcmurtry | Spider mites: their biology, natural enemies and control[END_REF] and infestations have been reported in Spain, Greece, Cuba, Morocco, and in Florida in the United States [START_REF] Ramos | Desarollo y reproducción de Polyphagotarsonemus latus (Acariformes: Tarsonemidae) sobre naranjo Valencia y limón (verdadero) en relación con lima persa[END_REF]Cáceres & Childers 1991;[START_REF] Tsagkarakou | Population structure in the spider mite Tetranychus urticae on Crete based on multiple allozymes[END_REF]Allam et al. 1999;[START_REF] Miranda | Application of the multiple correspondence analysis to the exploratory analysis of population data of the Tetranychus urticae (Koch) (Acari: Tetranychidae) and its predators. Influence of temperature, relative humidity and rainfalls[END_REF]. In the Mediterranean region, damages have decreased the value of commercial fruits [START_REF] Jacas | Side-effects of pesticides on selected natural enemies occurring in Citrus in Spain[END_REF][START_REF] Aucejo | Evaluation of the effectiveness of some miticides against Tetranychus urticae Koch (Tetranychidae) in clementines[END_REF][START_REF] Urbaneja | Efficacy of five selected acaricides against Tetranychus urticae (Acari: Tetranychidae) and their side effects on relevant natural enemies occurring in citrus orchards[END_REF].

In Europe, T. urticae is regarded as one of the main pests attacking grapevines [START_REF] Kassemeyer | Integrated Management of grapevine diseases and pests[END_REF] and its presence has been reported in France [START_REF] Rambier | Les tétranyques nuisibles à la vigne en France continentale[END_REF]), Spain (Arias Giralda & Nieto Calderón 1982), Switzerland [START_REF] Baillod | Migrations, population estimates and injuriousness of the common yellow spider-mite, Tetranychus urticae Koch in viticulture in Wallis[END_REF], and the former Czechoslovakia [START_REF] Hluchy | Damage potencial of the phytophagous Eriophyidae and Tetranychidae mites (Acari: Tetrapodili: Prostigmata) in grapevine[END_REF].

Okra cultivation in India presented losses between 12% and 36% in fruit production (Prasad & Singh 2007) and, in Iran, caused losses of 13% to 70% in the weight of fruits (Anaraki et al. 2007). Damage to tomatoes has been outlined by various authors [START_REF] Barbosa | As pragas do tomateiro e seu controle[END_REF][START_REF] Minami | O Tomateiro[END_REF][START_REF] Flechtmann | Ácaros de importância agrícola[END_REF][START_REF] Gonçalves | Variation of 2-Tridecanone level in tomato plant leaflets and resistance to two mite species (Tetranychus sp.)[END_REF][START_REF] Aragão | Efeito de aleloquímicos em tricomas foliares de tomateiro na repelência a ácaro (Tetranychus urticae Koch.) em genótipos com teores contrastantes de 2-tridecanona[END_REF].

In the United States, T. urticae populations are responsible for reducing the amount of fruit in strawberry fields [START_REF] Sances | Spider mite can reduce strawberry yields[END_REF]. In Africa, production loss can reach 50% (Coates 1974 1 , cited by Wysoki 1985). In Brazil, the two-spotted spider mite is the main pest in strawberry fields in the states of São Paulo and Rio Grande do Sul, and is found especially during fruit-bearing periods and during harvests, rendering most of the harvest unviable, given the damage and suspected contamination from pesticides (Tanaka et al. 2000;Moraes & Braun 2002).

1 COATES, T.J.D. (1994). The influence of some natural enemies and pesticides on various populations of Tetranychus cinnabarinus (Boisduval), T. lombardinii Baker & Pritchard and T. ludeni Zacher (Acari: Tetranychidae) with aspects of their biologies. Entomology Memoir, 42. Department of Agricultural Technical Services, Republic of South Africa.

Pest control

Chemical methods are most common for controlling T. urticae and have been studied in detail (for example: [START_REF] Watanabe | Controle biológico do ácaro rajado com ácaros predadores fitoseídeos (Acari: Tetranychidae, Phytoseiidae) em culturas de pepino e morango[END_REF][START_REF] Heungens | Chemical control of the two spotted spider mite Tetranychus urticae Koch on Hedera helix 'Anneborgh[END_REF][START_REF] Botton | IPM on peachs in Brazil: actual situation and future trends[END_REF][START_REF] Kimura | Evaluation of acaricides against the twospotted spider mite, Tetranychus urticae and the European red mite, Panonychus ulmi, from apple orchads in Aomori Prefecture[END_REF][START_REF] Barros | Tetranychus urticae Koch, 1836 (Acari: Tetranychidae) spider mite biological disequilibrium after insecticide applications on cotton plants[END_REF]. Alternatively, conventional biological control using predator mites (Acari: Phytodeiidae) has been carried out in different countries [START_REF] Khalequzzaman | Predatory efficacy of Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae) on the two spotted spider mite Tetranychus urticae (Acari: Tetranychidae)[END_REF][START_REF] Ferguson | Combined use of predatory mites for biological control of Tetranychus urticae (Acari: tetranychidade) in commercial greenhouse cucumber[END_REF]Moraes & Flechtmann 2008). In Brazil, predator mites Neoseiulus californicus (McGregor, 1954) and P. macropilis (Banks, 1905) are being used to control T. urticae on fruitbearing plants (apple and strawberry), ornamental plants, and in greenhouse crops [START_REF] Monteiro | Efeito do manejo de plantas daninhas sobre Neosiulus californicus (Acari: Phytoseiidae) em pomar de macieira[END_REF][START_REF] Marchetti | Flutuação populacional de ácaros (Acari) na cultura do morango no Estado do Rio Grande do Sul[END_REF][START_REF] Ferla | Ácaros de importância para a região do Sul do Brasil[END_REF][START_REF] Marchetti | Resposta functional de Phytoseilus macropilis Banks a diferentes populacões de Tetranychus urticae Koch (Acari: Phytoseiidae e Tetranychidae)[END_REF][START_REF] Ferla | Ácaros predadores (Acari) associados à cultura do morango (Fragaria sp., Rosaceae) em plantas próximas no Estado do Rio Grande do Sul[END_REF][START_REF] Oliveira | A phytoseiid predator from the tropics as potencial biological control agent for the spider mite Tetranychus urticae Koch (Acari: Tetranychidade)[END_REF]. Biological control along with chemical methods where biotypes of predator mites resistant to certain pesticides are used has provided a promising alternative for T. urticae control in the United States, Ukraine, South Africa and Australia [START_REF] Pringle | Biological Control of Tetranichid mites in South African[END_REF][START_REF] Waite | Managing spider mites in field-grows strawberries using Phytoseiulus persimilis and "pest-in-first" techinich[END_REF]). In Brazil, similar studies have been conducted on the predators N. californicus and P. macropilis (Banks) [START_REF] Sato | Management of Tetranychus urticae (Tetranychidae) in strawberry fields using Neoseiulus californicus (Phytoseiidae) and acaricides[END_REF][START_REF] Sato | Seleções para resistência e suscetibilidade, detecção e monitoramento da resistência de Tetranychus urticae ao acaricida clorfenapir[END_REF][START_REF] Poletti | Compatibilidade de agrotóxicos com ácaros predadores Neoseiulus californicus (McGregor) e Phytoseiulus macropolis (Banks) (Acari: Phytoseiidae)[END_REF].

Biological traits of T urticae favouring its pest status

The reproduction of T. urticae occurs via facultative parthenogenesis. In the absence of sexual reproduction, females have the ability to reproduce asexually. The ovaries are undergo complete development producing only haploid males. Following the first copulations, females produce predominately diploid females, which lead to haplodiploidy, whereby males are produced by arrhenotokous parthenogenesis and females by sexual reproduction. Therefore, a single female can generate offspring [START_REF] Helle | Parthenogenesis, chromosomes and sex[END_REF]. In addition, the species has great plasticity to change and feed on different host plants, which has probably contributed to the success of the worldwide colonization.

The presence of biotypes

Biotypes mean populations of a given species that have particular biological characteristics. The term "host race" represents a differentiated form, which may or may not crossbreed, but that feeds on specific host plants. This term is often used in entomological research and the term biotype must take precedence over "host race" [START_REF] Futuyma | Biologia evolutiva. 2nd ed[END_REF]. Differences in plant damaging observed among populations of phytophagous organisms can be linked to geographic distribution or host plant selection. For example, the red form of T. urticae infests and causes damage to citrus orchards in the Mediterranean region, particularly in Spain [START_REF] Aucejo | Evaluation of the effectiveness of some miticides against Tetranychus urticae Koch (Tetranychidae) in clementines[END_REF]Ansaloni et al. 2006;Abad et al. 2006[START_REF] Urbaneja | Efficacy of five selected acaricides against Tetranychus urticae (Acari: Tetranychidae) and their side effects on relevant natural enemies occurring in citrus orchards[END_REF], that commercializing in natura fruit, especially oranges, tangerines, and lemons [START_REF] Neves | Estudo da compatibilidade de cadeias integradas no Brasil: impacto das zonas de livre comércio -cadeia Citrus[END_REF]. However, T. urticae has not been reported damaging Brazilian citrus orchards. The reason why this species, which harms various hosts in Brazil, has not been found damaging citrus in the country remains unknown. This is an relevant example on the importante of studies on population genetic structure and population differenciation to address agricultural issues.

Another important factor in investigating genetic differences among T. urticae populations is the development of resistance to insecticides and miticides that results from excessive use of these products in attempts to control mites on crops.

The development of resistance to pesticides in T. urticae populations can provide the emergence of new biotypes that embarrass control and frustrate attempts to adopt Integrated Pest Management practices.

The resistance of T. urticae to pesticides has been documented in different crops in several countries (Cranham & Helle 1985;[START_REF] Miller | Effect of early season immigration on cyhexatin and fometanate resistance of Tetranychus urticae (Acari: Tetranychidae) on strawberry in central California[END_REF][START_REF] Edge | Organo-tin resistance in two-spotted spider mite, Tetranychus urticae Koch (Acarina: Tetranychidae) in Australia[END_REF][START_REF] Dennehy | Laboratory and field investigations of spider mite (Acari: Tetranychidae) resistance to the selective acaricide propargite[END_REF][START_REF] Grafton-Cardwell | Spider mite species (Acari: Tetranychidae) response to propargite basis for an acaricide resistance management program[END_REF][START_REF] Flexner | Field reversion of organotin resistance in twospotted spider mite following relaxition of selection pressure[END_REF]Tian et al. 1992;[START_REF] Landeros | Susceptibility and mechanism of resistance of Tetranychus urticae (Tetranychidae) populations from greenhouse roses[END_REF][START_REF] Lee | Acaricide resistance of Tetranychus urticae in apple orchads in Korea: implications for an acaricide resistance management[END_REF]). Croft & Hoyt (1983) 2 , cited by [START_REF] Prokopy | Apple insect pest management[END_REF], stated that T. urticae are the most difficult and burdensome pest to control in apple orchards in North America, due to their capacity to develop resistance to insecticides. In Brazilian apple orchards, T. urticae is not one of the main pests. In Brazil, problems with resistant T. urticae population to pesticides have been reported in cotton plants, rose bushes, strawberry fields, and peach trees [START_REF] Chiavegato | Resistência do ácaro rajado Tetranychus (T.) urticae (Koch, 1836) (Acari: Tetranychidae) proveniente de diferentes regiões algodoeiras aos acaricidas[END_REF]Suplicy Filho et al. 1994;[START_REF] Sato | Gene flow amosng Tetranychus urticae (Acari: Tetranychidae) populations in Greece[END_REF][START_REF] Sato | Resistência de Tetranychus urticae (Koch,1836) a acaricidas, em pessegueiro (Prunus Persica (L.) Batch) em Paranapanema e Jundiaí[END_REF][START_REF] Sato | Propargite resistance in Tetranychus urticae Koch (Acari: Tetranychidae) in the state of São Paulo, Brazil[END_REF][START_REF] Sato | Selections for fenpyroximate resitance and susceptibility, and inheritance, crossresistance and stability of fenpyroximate resistance in Tetranychus urticae Koch (Acari: Tetranychidae)[END_REF][START_REF] Sato | Seleções para resistência e suscetibilidade, detecção e monitoramento da resistência de Tetranychus urticae ao acaricida clorfenapir[END_REF][START_REF] Flores | Estabilidade, seleção e padrão de herança da resistência do ácaro rajado, Tetranychus uricae Koch (Acari: Tetranychuidae), ao Abamectin[END_REF]. Nevertheless, the introduction of resistant populations to insecticides or aggressive T. urticae populations would compromise integrated pest management programs currently used for orchards in the south of the country and would cause negative impacts on the production chain of Brazilian apples.

The risk of introducing and establishing new T. urticae biotypes that can possibly worsen economic problems in the country by importing plant products must be taken into account [START_REF] Ferreira | Subsídios para a quarentena vegetal. I. avaliação da importância quarentenária de ácaros associados a culturas frutíferas para o Brasil. II. disseminação de ácaros fitófagos através de material para propagação vegetativa de Arachis pintoi (Krap. & Greg.) (Fabaceae). Dissertação Mestrado. Escola Superior de Agricultura "Luiz de Queiroz[END_REF]. Studies on genetic variability in Brazilian T. urticae populations can help distinguish between populations already present in the country and introduced biotypes.

The use of molecular markers in Acarology

Choosing genetic markers with the adequate variability to the taxonomical level (e.g. genus, species, populations) to be examined is the first step to any study based on genetic differenciation. For istance, when comparing populations, genetic markers must be highly polymorphic to allow the detection of individual movements, which might be minimal. It is necessary to be familiar with the general properties, the fields of application, and the limits of each marker in order to ensure a choice that is compatible with the research objectives.

Studies with molecular markers can aid in understanding systematics, phylogenetics, and population structures in Acarology, and molecular techniques are becoming more and more used tools for taxonomists and acarologists in general.

They help not only in separating related species but also differentiating between subspecific categories and characterizing biotypes, thereby benefitting researchers involved in phytosanitary defence, biological control, parasitology, population biology, molecular ecology, control, and various other areas.

The first studies using molecular markers in acarology began in the 1990s [START_REF] Kaliszewski | The polymerase cham reaction and sequencing of mite DNA[END_REF][START_REF] Navajas | Intraspecific diversity of the cassava green mite Mononychellus progressivus using comparisons of mitochondrial and nuclear ribosomal DNA sequences and cross-breeding[END_REF][START_REF] Mozes-Koch | Separation of acarine species by enzymatic, immunological and molecular methods[END_REF]. Since then, reviews on the use of molecular markers in systematic and phylogenetic studies of mites comprising information on the methods, the main results, and perspectives have been published by [START_REF] Navajas | The application of molecular markers in the study of diversity in acarology: a review[END_REF]; Navajas (2001), [START_REF] Cruickshank | Molecular markers for the phylogenetics of mites and ticks[END_REF] and [START_REF] Navajas | Molecular diagnosis[END_REF].

Mites species of agricultural importance and displaying great diversity, have been the focus of studies using molecular techniques. These mainly belong to the Tetranychidae and Eriophyidae phytophagous families, and to the Phytoseiidae predator mites (Navajas et al. 1992(Navajas et al. , 1998(Navajas et al. , 1999(Navajas et al. , 2000;;Tsagkarakou et al. 1999;[START_REF] Navajas | The application of molecular markers in the study of diversity in acarology: a review[END_REF][START_REF] Noronha | Caracterização molecular de populações de Euseius citrifolius Denmark & Muma e Euseius concordis (Chant) (Acari: Phytoseiidae)) utilizando o seqüenciamento das regiões ITS1 e ITS2[END_REF]Tixier et al. 2006, Navajas & Navia 2010).

As for many other arthropods, the most commonly used markers in molecular systematics of mites are mitochondrial DNA (mtDNA) and nuclear ribosomal DNA (rDNA). They differ considerably in terms of genomic organization (in that one is of mitochondrial and the other of nuclear), evolution mode (they present differential rates of evolution) and transmission (maternal and biparental transmission, respectively). Given the importance and utility of mtDNA and rDNA markers in Acarological research, their general characteristics, use, as well as the most important subjects related to them, have been revised.

Phytophagous mites -nuclear ribosomal DNA and mitochondrial DNA

Phylogenetic studies seek to reconstruct the evolutionary history of groups of organisms (Amorim 1994). Recent advances in population genetic theories along with rapid advances in molecular techniques have allowed access to detailed phylogenetic information on populations, in which different haplotypes represent operational taxonomic units (OTU) [START_REF] Crandall | Applications of intraspecific phylogenetics[END_REF][START_REF] Harvey | What this book is about[END_REF]. DNA sequences can be important tools in reconstructing evolutionary relationships among organisms [START_REF] Weekers | Phylogenetic relationship inferred from ribosomal ITS sequences and biogeografhic patterns in representatives of the genus Calopterix (Insecta: Odonata) of the West Mediterranean and adjacent west european zone[END_REF]).

However, it is widely admitted that the phylogenetic content of a single genetic marker gives only a partial view of the phylogeny and the haplotype ancestry reconstruction of the species.. It is then important to consider the way in which the contrasting properties of markers can affect their capacities to construct phylogenies (Navajas & Boursot 2003), and that phylogenetic studies based on the results of a single molecular marker are unreliable and can lead to unfounded conclusions (Brower et al. 1996;[START_REF] Ross | Assessing genetic structure with multiple classes of molecular markers: a case study involving the introduced fire ant Solenopsis invicta[END_REF]Navajas & Boursot 2003). For example, phylogenetic analyses using sequences of cytochrome oxidase 1 regions (COI) of mtDNA and the internal transcribed spacer (ITS2) of rDNA of two closely related species of the genus Tetranychus, T. urticae and T. turkestani, showed that these two species are polyphyletic for mtDNA while monophyletic for rDNA. The nucleotide diversity was extensive for the mtDNA COI gene, 3-4%, and extremely low for the ITS2 region at less than 0.5%. The results indicated that, despite biparental transmission and multiplicity of copies in the genome, nuclear rDNA had a smaller effective population size than mtDNA in these species (Navajas & Boursot 2003).

The authors argue that the combination of efficient concerted evolution and/or gene conversion in the rDNA cluster, the haplodiploidy of these species and the femalebiased sex ratio could account for this apparent contradiction in relation to the expected pattern. Likewise, the the phylogenetic contect of these to markers was contrasted when comparinig the the phylogeny of Japanese species in the Panonychus Yokoyama genus, based on sequences of the COI region (mtDNA), and those obtained using regions of the rDNA (Toda et al. 2000).

In general, the mutation rate of mtDNA sequences tend to be higher that those of nuclear genes and are appropriate for population studies. Because of the haploid nature of mitochondrial genes, the frequency of a single mtDNA haplotype within a population should fluctuate more rapidly than the frequencies of nuclear DNA. Thus, mtDNA should be more sensitive to founder events and small population sizes than nuclear DNA. Consequently, the probable loss or gaining of a mtDNA haplotype will be significant for small populations and only a small migration among populations would be sufficient to fix a mtDNA lineage in all populations (Roderick 1996). Navajas et al. (1998) noted in T. urticae populations with different origins sampled in a wide geographic area, approximately 5% divergence of nucleotides in the COI region of the mtDNA, while no variation was found in the ITS2 region. The authors also compared the evolution rates of these two markers in one group of species of the same genus and did not obtain the same results, realizing that in other Tetranychus species, the ITS2 region also varied. The results indicated that the ITS region evolved 2.5 times faster than the COI region in Tetranychus species. The authors suggested that the homogeneity of ITS2 sequences in T. urticae was due to its capacity to colonize and consequently, to its relatively high gene flow.

The utility of these markers in tracing invasive species was shown in a study on Mononychellus progressivus Doreste [START_REF] Navajas | Intraspecific diversity of the cassava green mite Mononychellus progressivus using comparisons of mitochondrial and nuclear ribosomal DNA sequences and cross-breeding[END_REF]. The genetic structure of this invasive mite native to the Neotropical region and accidentally introduced to East Africa in the 1970s, was studied using COI and ITS2 sequences. The results suggested that M. progressivus was introduced from populations originating in Colombia [START_REF] Navajas | Intraspecific diversity of the cassava green mite Mononychellus progressivus using comparisons of mitochondrial and nuclear ribosomal DNA sequences and cross-breeding[END_REF]. This is relevant information to guide the search in the native are of the pest of adecuate biological control agents to be introduced in Africa.

Likewise, based on the analyses of ribosomal DNA sequences (ITS) and mitochondrial DNA sequences (16S and COI), Navia et al. (2005) studied the phylogeography of Aceria guerreronis Keifer (Eriophyidae) populations worldwide, and suggested that the species originated in America and determined the introduction routes in Africa/Asia.

Another important use of molecular data in Acarogly is to address taxonomic issues. The genus Amphytetranychus Oudemans was restored based on the convergence of molecular (ITS2 and COI) and morphological data (Navajas et al. 1997). The molecular approach was also useful to propose the species status of T. pueraricola (Ehara & Gotoh) by discriminating it from T. urticae Koch (Navajas et al. 1998). Navajas et al. (2001) established the synonymy between T. kanzawai Kishida and T. hydrangea Pritchard & Baker, based on analyses of the ITS2 sequences and on crossbreeding experiments. Intraspescific variation was reported in T. kanzawai in studies with ITS and COI sequences (Hinomoto & Takafuji 2001).

Genetic data to study ecological differenciation in Tetranychidae

Relationships between phylogeny and geographic distribution represent the phylogeographic patterns for a given species. In addition, environemental conditions might select specific genotypes adapted to specific a habitat which drives population differenciation.

The genetic structure of a species can be inferred indirectly through genetic data. This data can be analyzed in two distinct ways: 1) populations are treated as genetic units and measurements of similarity among populations are estimated, and 2) phylogeographic analysis, in which relationships between genotypes (phylogenies of alleles or haplotypes) of one or more populations can be investigated according to its geographic location. Variations in DNA sequences are analyzed to establish relations between haplotypes of the same loco (Roderick 1996).

In Europe and Asia, genetic differentiation among T. urticae populations has been studied using genetic markers, together with the evaluation of biological, behavioural and morphological parameters [START_REF] Gotoh | Host race formation in Tetranychus urticae: genetic differentiation, host plant preference, and mate choice in a tomato and a cucumber strain[END_REF][START_REF] Hence | The use of fecundity, lobe biometry and the RAPD-PCR technique in order to compare strains of Tetranychus sp[END_REF]Navajas 1998;Tsagkarakou et al. 1999;Navajas et al. 2000). The results indicate that the variability of the species can be linked to geographic distribution but also to host plant uses. This point has been the object of broad discussions regarding this species. [START_REF] Gotoh | Host race formation in Tetranychus urticae: genetic differentiation, host plant preference, and mate choice in a tomato and a cucumber strain[END_REF] studied the formation of host races in T. urticae by estimating genetic differences, host plant preference, mating selection in lineages collected from pumpkins and tomatoes, and found two host races that were biologically isolated in sympatry. [START_REF] Tsagkarakou | Population structure in the spider mite Tetranychus urticae on Crete based on multiple allozymes[END_REF] did not find differences between T. urticae populations collected on different hosts when the selected plants fell within a range of 50 meters. But when the distance was above 150 meters, differentiation was noted. Studies of T. urticae performed on open-field citrus orchards in the Mediterranean region indicated a greater genetic similarity between populations collected from citrus trees in different regions than between those collected from different hosts of a same region [START_REF] Tsagkarakou | Gene flow amosng Tetranychus urticae (Acari: Tetranychidae) populations in Greece[END_REF].

While the study of the host plant in Tetranychus species has frecuently studied, the weight of this factor in driving population differenciation should be weighted by the effect of geographical distance which tend to limit gene flow among populations just creating physical barriers to migration. Some attemps to evaluate the role of host plants and the geographic distance in T. urticae show that the later tend to be a strong factor in the process of genetic differentiation (Navajas, et al. 1998;Tsagkarakou et al. 1999;Navajas et al. 2000).

Using COI sequences two distinc lineages of T. urticae were detected throughout an extensive part of its distribution area. One of the lineages gave evidence of a long-standing colonization in the Mediterranean region; while the otherof a more recent colonization in temperate regions of the Northern hemisphere (Navajas 1998). But any association to the host plant stood out at a continental geographical scale.

Tetranychus urticae mites have a cospolitant distribution, they are present in a wide range of habitats and have numerous plant hosts in Brazil. Studies using molecular markers and basic phylogenetic and phylogeographic research are useful tools for characterizing populations, helping to determine whether biotypes are present or not. In turn, these results can supplement decisions on how to manage this mite as well as to identify populations those pose a risk to Brazil and that must be contained during quarantine inspections.

Methods in molecular phylogenetic analysis

Whilst studying the evolution of species, is necessary to look for homologies or similarities inherited from common ancestry among haplotypes of the species involved, with the aim of finding an evolutionary model that assumes that different species derive from a common ancestor. Comparative sequence analysis is an important tool in genetics and uses phylogenetic trees to describe the evolutionary relationship between the sequences of nucleotides that emerge from extracting DNA.

The process of making phylogenetic inferences has four basic steps [START_REF] Holder | Phylogeny estimation: traditional and Bayesian approachs[END_REF]: 1) collect sequences for comparison; 2) align sequences (pairing the bases of one sequence with those of homologous sequences); 3) choose an evolution model of the sequences (generally, software is used to test various models and infer those which best adapt to the sequences under analysis); 4) create a set of phylogenetic trees based upon the sequences being analyzed and the evolution models chosen (using phylogenetic methods of reconstruction by searching for multiple trees followed by statistical significance tests: e.g. "bootstrap" or the algorithm "Markov chain Monte Carlo" -MCMC). This set of trees can be summarized using "consensus trees" techniques, which show the parts of the trees that are found in most or all of the trees.

For aligning the DNA sequences of different species, the hypothesis of the homology between the bases (or nucleotides) is assumed. Among the aligning sequences programs, Clustal W (Thompson, 1994) is a basic tool that produces multiple alignments of divergent sequences (DNA or protein), which aligns sequences so that identities, similarities and differences can be noticed and calculates the best score. The evolutionary differences can be viewed in cladograms or phylograms.

In this way, traditional phylogenetic reconstruction methods provide a series of possible phylogenetic trees based on the sequences analyzed. The statistical bootstrap method, which establishes confidence intervals in the phylogenies [START_REF] Felsenstein | Confidence limits on phylogenies: An approach using the bootstrap[END_REF], provides information on the robustness of each ramification. Due to its versatility, this method has traditionally been the most used in molecular phylogeny. A clear presentation on the bootstrap method can be found in Solignac et al. (1995). In general, the bootstrap method provides information on the parts of the tree that provide either low or high support. The exact interpretation of the bootstrap value is relative. Certainly, the higher the bootstrap value obtained the better.

According to [START_REF] Holder | Phylogeny estimation: traditional and Bayesian approachs[END_REF], high bootstrap proportions are necessary conditions, but not enough to ensure high reliability in a group.

The methods traditionally used in phylogenetic reconstructions are: algorithm NJ (Neighbour-Joining) and trees, which use optimization criteria such as maximum parsimony (MP) or maximum likelihood (ML). The principles of each of these methods, as well as the advantages and disadvantages, are presented by Solignac et al. (1995) and [START_REF] Holder | Phylogeny estimation: traditional and Bayesian approachs[END_REF]. More recently, some new analysis methods bring new perspectives to evolutionary analysis, like the Bayesiana approach [START_REF] Huelsenbeck | Bayesian inference of phylogeny and its impact on evolutionary biology[END_REF][START_REF] Holder | Phylogeny estimation: traditional and Bayesian approachs[END_REF], which support statistical conclusions based on coalescent method and the algorithm Markov Chain Mont Carlo (MCMC). An useful discussion on these methods are in [START_REF] Luikart | Statistical analysis of microsatellite DNA data[END_REF] and [START_REF] Holder | Phylogeny estimation: traditional and Bayesian approachs[END_REF]. 

Introduction

The Tetranychidae or spider mites comprise a large group of phytophagous mites, with many of its species considered as important pests of agricultural crops worldwide. So far, this family includes about 1250 species, distributed in 74 genera;

182 tetranychid species have been recorded in South America and 104 species in Brazil (Migeon and Dorkeld, 2008;Bolland et al., 1998).

The genus Monoceronychus McGregor, 1945 (Bryobiinae, Hystrichonychini) includes19 species (Migeon and Dorkeld, 2008;Bolland et al., 1998) collected from monocotyledonous plants, mainly on grasses (Poaceae). Most were described from North America, 15 from the United States (Pritchard & Baker, 1955;[START_REF] Krantz | Monoceronychus boreus, a new species of spider mite from Oregon[END_REF][START_REF] Tuttle | The spider mites of Arizona (Acarina: Tetranychidae)[END_REF]Tuttle & Baker, 1968;[START_REF] Baker | New species and further notes on the Tetranychoidea mostly from the south-western United States (Acarina: Tetranychidae and Tenuipalpidae)[END_REF] and two from Mexico [START_REF] Tuttle | Reports of Tetranychus urticae on Parthenium sp were made in India (Gupta and Gupta, 1994). In the Americas, the first report of its presence on Parthenium sp. occurred in Rio[END_REF]; the remaining species are from Africa (South Africa) (Meyer, 1974) and South America (Brazil). Monoceronychus bambusicola Feres & Flechtmann, 1995 was described from Bambusa from Brazil and was recently recorded from Japan (Gotho & Shida, 2007). Monoceronychus linki Pritchard & Baker, 1955 is the other species found in Brazil (Flechtmann & Baker, 1975) also found in Paraguay [START_REF] Aranda | A report on the Tetranychidae of Paraguay[END_REF]) and Cuba [START_REF] Almaguel | Nuevo acaro Tetranychidae en cana (Saccharum officinarum)[END_REF].

The genus Oligonychus Berlese, 1886 (Tetranychinae, Tetranychini) contains 203 valid species, 52 of which have been recorded from South America and 25 from Brazil. Twelve species of Oligonychus were collected from grape worldwide, six from Brazil: O. anonae Paschoal, 1970, O. biharensis (Hirst, 1925), O. coffeae (Nietner, 1861), O. mangiferus (Rahman & Sapra, 1940), O. punicae [START_REF] Hirst | Descriptions of new mites including four new species of red spider[END_REF], O. yothersi, [START_REF] Mcgregor | Four new tetranychids[END_REF]) (Migeon and Dorkeld, 2008;Bolland et al., 1998).

The genus Eotetranychus Oudemans, 1931, has 185 valid species, 18 of which were recorded from South America and eight from Brazil. Two Eotetranychus species were reported from rose in South America, E. deflexus (McGregor, 1950) and E. lewisi (McGregor, 1943).

In (Malufi, 2000). The annual average temperature is 19.6°C, the maximum is 30.8°C in January and the mi nimum is 9.2°C in July (Tempo Agora, 2009). The host plant, E. distichophylla, is a perennial grass described from Argentina and its present distribution is South and North America [START_REF] Soreng | Catalogue of new world grasses (Poaceae)[END_REF].

The local climate of the collection site of the Oligonychus specimens - showing a particular arched pattern of granulation. Setae h1 longer than in female, more than five times longer than f1.

Leg chaetotaxy, from coxa to tarsus (solenidia in parentheses). There were two observations of alternative setal counts.

I . 2-1-6-2-6-9(1) + 2 duplex II. 2-0-3-2-4-7 + 1 duplex III. 1-1-2-1-4-7 or 6
IV. 1-0-2-1-4-7 or 6 Paratypes. Five females, two males and 10 protonymphs with same host and data as holotype.

Etymology:

The name "tche" used in the first part of the specific designation was based on a typical word of the Rio Grande do Sul state, Brazil, where the mite was collected. The source of the name "tche" is uncertain, but is usually employed to show surprise. The last part, "-ensis" comes from Latin and means "of" or "belonging to". Thus, tchecensis means "of" or "relating to" "a surprise", in the vernacular of the Rio Grande do Sul region.

Remarks:

The new species is close M. linki Pritchard &Baker, 1955 andM. bambusicola, Feres &Flechtmann, 1995, in Paratypes. Three females and four males, same host and collection data as holotype.

Etymology:

The specific designation is a composite of two Greek words. The Greek prefix filo means friend; Greek suffix eno means wine. Thus, fileno means a "friend of wine" and refers to the typical beverage produced from grape, the host plant of the new species. 

Remarks

Introduction

The Tetranychidae Donnadieu family includes a large number of strictly phytophagous mites; with many of its species considered as significant crop pests to worldwide agriculture (Jeppson et al. 1975;[START_REF] Helle | Spider mites: their biology, narural enemies and control[END_REF].

The number of described tetranychids remained stable for 75 years and then increased when their economic incidence for agriculture became more significant (Bolland et al. 1998) In Brazil, taxonomic studies on the Tetranychidae were initially conducted between 1920 and 1930. Some of the first reports substantiate the Tetranychus gloveri [START_REF] Banks | The red spiders of the United States (Tetranychus and Stigmaeus)[END_REF], and describe the Mononychellus tanajoa Bondar, 1938, based on specimens collected in Brazil by G. Bondar, a Russian researcher living in Bahia [START_REF] Bondar | Insetos daninhos e moléstias dos feijões na Bahia[END_REF][START_REF] Bondar | Notas entomológicas de Bahia III[END_REF]. In the 1960s numerous contributions were made by pioneers in agricultural mite studies in Brazil, such as C. H. W. Flechtmann (Flechtmann 1967(Flechtmann , 1967a(Flechtmann , 1967b[START_REF] Flechtmann | Tetranychus (T.) urticae Koch. 1836 (Acari) praga severa de orquídeas (Catasetum sp.) em ripados[END_REF](Flechtmann , 1975[START_REF] Flechtmann | A report on the Tetranychidae (Acari) of Brazil: an emmendation[END_REF]Flechtmann andBaker 1970, 1975) and A. D.

Paschoal (ESALQ, USP, Piracicaba) (Paschoal 1970(Paschoal , 1970a)), followed by R. J. F.

Feres (UNESP, São José do Rio Preto São Paulo) [START_REF] Feres | Dois Novos Eotetranychus (Acari, Tetranychidae) do Estado de São Paulo[END_REF][START_REF] Feres | Allonychus brevipenis, a new species from Triplaris surinamensis Cham. in Brazil with a new host records for A. brasiliensis (McGregor) and A. reisi Paschoal (Acari: Tetranychidae)[END_REF], Feres and Flechtmann 1986, 1986a, 1988[START_REF] Futuyma | Biologia evolutiva. 2nd ed[END_REF], 1995a, 1995b). From then on these and other new researchers have been conducting important studies on tetranychids in several fields such as taxonomy, ecology, biology, control and resistance. This brought irrefutable advances to scientific knowledge of this group of mites in the country.

Currently 185 of the 1,257 described species are reported in South America and 104 are in Brazil (Bolland et al. 1998;Migeon and Dorkeld 2006).

The most intensive search for Tetranychidae mites in Brazil, the largest country in South America, has been centered in the southeast region and is usually associated with economically important crops. Agriculture has expanded toward new regions in the country and even coming close to biological reserves. It is important to continue researching tetranychids in previously unexplored areas and adding information that would increase our understanding of this family. This paper presents new information about the occurrence and distribution of Tetranychidae in five Brazilian regions and in Uruguay, found on plants ranging from ornamental, fruitbearing, vegetable, grass, oilseed, and large crops species. New hosts and localities for tetranychid mites in Brazil, South America, and the world are reported.

Material and methods

A legal permit to collect zoological material for scientific purposes is required by the Brazilian Institute for the Environment and Renewable Natural Resources (IBAMA) and therefore, we requested and were issued permit number 231/2006 CGFAU/IBAMA.

Mite collection

Mites were collected between October 2004 and July 2008. A total of 550 samples were collected from several host plants found in five regions in Brazil that include the states of Acre, Bahia, Ceará, Espírito Santo, Goiás, Minas Gerais, Mato Grosso do Sul, Paraná, Pernambuco, Rio Grande do Norte, Rio Grande do Sul, Roraima, Santa Catarina, São Paulo, Sergipe, and the Federal District (Fig. 1; Supplementary Material, Appendix 1). One additional collection was conducted outside Brazil in Uruguay. Plants were collected when they presented symptoms of tetranychid attacks, according to [START_REF] Moraes | Manual de acarologia -Acarologia básica e ácaros de plantas cultivadas no Brasil[END_REF]. The geographical points of collection were logged into a Garmin 12 GPS using WGS84 (World Geodetic System 84) datum, and maps were created using the ARCGIS 9.0 program (ESRI Company).

Inspecting and processing plant samples

Mites were collected from the plants using the washing/sieving extraction method [START_REF] Miranda | Brevipalpus mites Donnadieu (Prostigmata: Tenuipalpidae) associated with ornamental plants in Distrito Federal, Brazil[END_REF]). The resulting 70% ethanol solution was inspected under a stereomicroscope (Zeiss -Stemi SV 6 and Olympus SZx122) connected to a transilluminator (Highlight 3000-1) at 50x magnification. Finally, Tetranychidae mites were directly mounted on microscopic slide preparations in Hoyer's medium. From each population, 25 females (dorso-ventral position) and ten isolated males (lateral position) were mounted to identify the species by morphological analysis. When only a few males were present in a sample, all of them were slide-mounted.

Identifying mite species

Microscopic slide preparations of specimens were examined under phase contrast and interference microscopes in 40x and 100x objectives (Leitz Wetzlar and Nikon Eclipse 80i). Morphological identification was conducted by examining females and males relevant taxonomic characteristics of Tetranychidae systematics. The shape of the aedeagus (male genitalia) was used to classify the species according to pertinent literature (Pritchard and Baker 1955, Meyer 1974, 1987, Baker and Tuttle 1994, Ehara, 1999), which were then compared to specimens that had been deposited in the Reference Mite Collection of the Laboratory of Plant Quarantine, Embrapa Genetic Resources and Biotechnology, Brasília, Brazil. The studied material was also deposited in this collection as voucher specimens. deposited in the Reference Mite Collection of the Laboratory of Plant Quarantine, Embrapa Genetic Resources and Biotechnology, Brasília, Brazil. The studied material was also deposited in this collection as voucher specimens.

Results and discussion

Tetranychid mites were found on 37.1% (n=204) of the 550 plant material samples that were collected (Supplementary Material, Appendix 1). Twenty-one species of Tetranychinae and one of Bryobiinae were identified on the 204 samples from different localities and inspected hosts (Table 1).

Several samples contained more than one species of tetranychids on the aff. mangiferus and O. fileno were found on grapes in the municipality of Pirapora. Foott (1962Foott ( , 1963) ) reported phytophagous mite species coexisting on a single host.

The fact that different species coexist on a single host requires careful sampling and collecting larger numbers of specimens in microscopic preparations. This allows for greater representation of mite fauna and increases the probability of identifying all

Tetranychidae species present on a specific host and/or locality.

New hosts for Tetranychidae mites in Brazil, South America and the world

New hosts were found for 11 tetranychid mite species: E. banksi, M. tanajoa, O.

anonae, O. mangiferus, T. bastosi, T. desertorum, T. evansi, T. ludeni, T. mexicanus, T. Neocaledonicus, and T. urticae (Table 2). The new hosts are listed below according to mite species and host plant family. * The columns do not equal a total of 207 collections due to repeated hosts in some localities

These are the first reports of E. banksi on Solanaceae in the world.

Tetranychinae Berlese, Tetranychini Reck

Mononychellus tanajoa [START_REF] Bondar | Notas entomológicas de Bahia III[END_REF] Tetranychus tanajoa [START_REF] Bondar | Notas entomológicas de Bahia III[END_REF]. Type-host: Manihot ultilissima and M. aipim.

Type-locality: Bahia, Brazil. This is the first report of O. mangiferus on castor oil plants and pomegranates in the Neotropics. This mite was previously found on these host plants in India (Gupta 1976, Gupta and[START_REF] Gupta | lunatus and M. atropurpureum are new hosts for T. urticae in South America. Passifloraceae Passiflora edulis Sims (yellow passion fruit), Embrapa Roraima, Boa Vista, Roraima, 17.V.2007. Tetranychus urticae on passion fruit (P. caerulea L. and P. edulis ) was reported in Venezuela (Doreste 1967) and in Australia (Passiflora sp.) (Gutierrez and Schicha 1983). This is the first report of T. urticae on passion fruit (P. edulis) in Brazil. Poaceae Triticum aestivum L. (wheat), Embrapa Trigo, Passo Fundo[END_REF]. Mussaenda L. is reported for the first time as a host for O. mangiferus.

Fabaceae

Tetranychus bastosi [START_REF] Tuttle | Spider mites (Tetranychidae: Acarina) of the state of Ceará, Brazil[END_REF] Tetranychus (Tetranychus) bastosi [START_REF] Tuttle | Spider mites (Tetranychidae: Acarina) of the state of Ceará, Brazil[END_REF] Host: Morus rubra L. Type-locality: Crato, Brazil.

Malvaceae

Gossypium barbadense L. (wild cotton), Empresa de Pesquisa Agropecuária de

Minas Gerais -EPAMIG, Nova Porteirinha, Minas Gerais, 10.IV.2006. This is first report of T. bastosi on cotton (G. barbadense). This specie was observed previously on the Malva rotundifolia L. [START_REF] Tuttle | Spider mites (Tetranychidae: Acarina) of the state of Ceará, Brazil[END_REF].

Tetranychus desertorum [START_REF] Banks | The red spiders of the United States (Tetranychus and Stigmaeus)[END_REF] Tetranychus desertorum Banks, 1900. Type-host [START_REF] Ehara | Notes on some tetranychid mites of Japan[END_REF]. Infestations of T.

desertorum in Brazil were registered on Acnistus cauliflorus (Flechtmann 1967);

Brugmansia suaveolens [START_REF] Furtado | Search for effective natural enemies of Tetranychus evansi in south and southeast Brazil[END_REF]; Brunfelsia sp, [START_REF] Flechtmann | Two new plant feeding mites from Brachiaria ruziziensis in citrus groves in São Paulo, Brazil and new distribution records of other plant mites in Brazil[END_REF];

Lycopersicon esculentum (Flechtmann 1967) and Solanum tuberosum (Paschoal 1970a).

Solanum gilo is reported as the new host for T. desertorum.

Tetranychus evansi [START_REF] Baker | The tetranychoid mites of Africa[END_REF] Tetranychus evansi [START_REF] Baker | The tetranychoid mites of Africa[END_REF]. Type-host: Lycopersicon esculentum L. Type-locality: Mauritius (Indian Ocean).

Caricaceae

Carica papaya L. (papaya), Mumbecas farm, Recife, Pernambuco, 11.VIII.2006.

Tetranychus evansi was originally described using samples collected from tomato plants [START_REF] Baker | The tetranychoid mites of Africa[END_REF]. However, this mite had already been discovered in Brazil by [START_REF] Silva | Um novo ácaro nocivo ao tomateiro na Bahia[END_REF] who described it as T. marianae McGregor.

Since then it has been reported in the United States (Bolland et al. 1998) and more recently in Africa, Argentina, China, Spain, France, Greece, Israel, Italy, Puerto Rico, Portugal, and Taiwan (Migeon and Dorkeld 2006). Tetranychus evansi is currently reported on 93 host species (Migeon and Dorkeld 2006), most of which belong to the Solanaceae family (Bolland et al. 1998;Moraes et al. 1987). In Europe [START_REF] Ferragut | Tetranychus evansi Baker & Pritchard (Acari, Tetranychidae), una nueva araña roja en los cultivos horticolas españoles[END_REF] reported infestations of this mite in several plant families. However, the only reports on the biology of the T. evansi are for those found on tomato plants.

Carica papaya is new host for the T. evansi.

Tetranychus ludeni Zacher, 1913

Tetranychus ludeni Zacher, 1913(Zacher 1913 Tetranychus ludeni was previously found on C. lanatus, P. hortorum and P.

domestica in Africa, Central America (El Salvador), Australia and India (Meyer and Ryke 1959;[START_REF] Davis | Studies of Queensland Tetranychidae. 3. Records of the genus Tetranychus[END_REF][START_REF] Andrews | Spider mites of El Salvador, Central America (Acari: Tetranychidae)[END_REF]Gutierrez and Schicha 1983;[START_REF] Gupta | lunatus and M. atropurpureum are new hosts for T. urticae in South America. Passifloraceae Passiflora edulis Sims (yellow passion fruit), Embrapa Roraima, Boa Vista, Roraima, 17.V.2007. Tetranychus urticae on passion fruit (P. caerulea L. and P. edulis ) was reported in Venezuela (Doreste 1967) and in Australia (Passiflora sp.) (Gutierrez and Schicha 1983). This is the first report of T. urticae on passion fruit (P. edulis) in Brazil. Poaceae Triticum aestivum L. (wheat), Embrapa Trigo, Passo Fundo[END_REF]. This is the first report of these hosts in South America.

Rutaceae

Citrus limonia L. (mandarin-lime), Universidade Federal de Lavras, Lavras, Minas Gerais, 09.V.2006. This is the first report of Citrus limonia as a host for the T. ludeni.

Tetranychus mexicanus (McGregor, 1950) Tetranychus mexicanus, (McGregor, 1950) neocaledonicus in the world. However, the presence of T. neocaledonicus on Cestrum sp. has been registered in the state of Rio de Janeiro [START_REF] Flechtmann | A small collection of spider mites from Jaguanum Island, Rio de Janeiro, Brazil, with description of Aponychus insularis (Prostigmata: Tetranychidae)[END_REF].

Other plant species belonging to the Ocimum and Morus genera (O. sanctum L., M. alba L., M. australis Poir, M. indica L., M. nigra L., M. sp.) were registered as hosts for T. neocaledonicus in Cuba, India, Japan and Thailand (Livshits and Salinas-Croche 1968;[START_REF] Baker | Plant-feeding mites of Thailand (Tetranychidae, Tenuipalpidae, and Tuckerellidae)[END_REF]Gupta 1992;[START_REF] Gupta | lunatus and M. atropurpureum are new hosts for T. urticae in South America. Passifloraceae Passiflora edulis Sims (yellow passion fruit), Embrapa Roraima, Boa Vista, Roraima, 17.V.2007. Tetranychus urticae on passion fruit (P. caerulea L. and P. edulis ) was reported in Venezuela (Doreste 1967) and in Australia (Passiflora sp.) (Gutierrez and Schicha 1983). This is the first report of T. urticae on passion fruit (P. edulis) in Brazil. Poaceae Triticum aestivum L. (wheat), Embrapa Trigo, Passo Fundo[END_REF][START_REF] Ehara | Discovery of Tetranychus neocaledonicus André (Acari: Tetrnychidae) from Amami-Oshima Island, Japan[END_REF]. Cases of Tetranychus urticae on Zingiberaceae (Curcuma longa L.) were reported in India [START_REF] Gupta | lunatus and M. atropurpureum are new hosts for T. urticae in South America. Passifloraceae Passiflora edulis Sims (yellow passion fruit), Embrapa Roraima, Boa Vista, Roraima, 17.V.2007. Tetranychus urticae on passion fruit (P. caerulea L. and P. edulis ) was reported in Venezuela (Doreste 1967) and in Australia (Passiflora sp.) (Gutierrez and Schicha 1983). This is the first report of T. urticae on passion fruit (P. edulis) in Brazil. Poaceae Triticum aestivum L. (wheat), Embrapa Trigo, Passo Fundo[END_REF]. This is the first report of T. urticae on alpinia (A. purpurata) in the world.

Tetranychus urticae

The samples collected in Uruguay revealed a new host for the spider mite (T.

urticae), as seen in the report below.

Verbenaceae

Lantana camara L. (wild sage), Rio Negro, San Javier, Uruguay, 06.VI.2007. (-58.1312 S; -32.6368 W -collected by D. Navia and E. Castiglione).

T. urticae was found on L. camara fin the United States and in India [START_REF] Thewke | The spider-mite complex (Acarina: Tetranychoidae) in Missouri[END_REF][START_REF] Gupta | lunatus and M. atropurpureum are new hosts for T. urticae in South America. Passifloraceae Passiflora edulis Sims (yellow passion fruit), Embrapa Roraima, Boa Vista, Roraima, 17.V.2007. Tetranychus urticae on passion fruit (P. caerulea L. and P. edulis ) was reported in Venezuela (Doreste 1967) and in Australia (Passiflora sp.) (Gutierrez and Schicha 1983). This is the first report of T. urticae on passion fruit (P. edulis) in Brazil. Poaceae Triticum aestivum L. (wheat), Embrapa Trigo, Passo Fundo[END_REF]. The Uruguay report of T. urticae on L.

camara is the first in South America.

New localities for Tetranychidae mites in Brazil

New localities have been registered for Eotetranychus tremae, O. anonae, T.

gloveri, P. ulmi, and E. smithi (Table 2).

Tetranychinae Berlese, Tetranychini Reck

Eotetranychus tremae [START_REF] Leon | Two new Eotetranychus and a new Oligonychus from Southern Florida (Acarina: Tetranychidae)[END_REF] Eotetranychus tremae De [START_REF] Leon | Two new Eotetranychus and a new Oligonychus from Southern Florida (Acarina: Tetranychidae)[END_REF]. Type-host: Trema floridanum. Type-locality:

Coral Gables, United States.

Reports of Eotetranychus tremae infesting the Acalypha sp. ornamental plant have been registered in Viçosa, Minas Gerais. Previous reports in Brazil were registered in the states of Rio de Janeiro [START_REF] Flechtmann | A small collection of spider mites from Jaguanum Island, Rio de Janeiro, Brazil, with description of Aponychus insularis (Prostigmata: Tetranychidae)[END_REF]) and São Paulo [START_REF] Flechtmann | Two new plant feeding mites from Brachiaria ruziziensis in citrus groves in São Paulo, Brazil and new distribution records of other plant mites in Brazil[END_REF][START_REF] Daud | Diversidade e flutuação populacional de ácaros (Acari) em Mabea fistulifera Mart. (Euphorbiaceae) de dois fragmentos de mata estacional semidecídua em São José do Rio Preto, SP[END_REF]Feres et. al. 2005).

Oligonychus anonae Paschoal, 1970 Oligonychus anonae Paschoal, 1970. Type-host: Annona muricata L., Persea americana Mill., Vitis vinifera L. Type-locality: Brazil. Paschoal (1970, 197a) Tetranychus gloveri [START_REF] Banks | The red spiders of the United States (Tetranychus and Stigmaeus)[END_REF] Tetranychus gloveri [START_REF] Banks | The red spiders of the United States (Tetranychus and Stigmaeus)[END_REF]. Type-host: Gossypium hirsutum L. Type-locality:

Baton Rouge, United States

Tetranychus gloveri was reported on beans in Bahia [START_REF] Bondar | Insetos daninhos e moléstias dos feijões na Bahia[END_REF] and on papaya (unspecified location) (Migeon and Dorkeld 2006). Reports of its presence on papaya (Carica papaya L.) in the state of Rio Grande do Norte indicate a new locality for this species.

Panonychus ulmi, (Koch, 1836)

Tetranychus ulmi, Koch, 1836. Koch (1836). Type-host: Ulmus sp. Type-locality:

Regensburg, Germany.

Reports of the European red mite (P. ulmi) on grape vines (Vitis vinifera L.)

were registered for the first time in Brazil in 2008 when damages caused by these mites were first noticed on crops in Rio Grande do Sul [START_REF] Ferla | Ocorrência do ácaro vermelho europeu associado à cultura da videira no Rio Grande do Sul, Brasil[END_REF]. In this work, a second occurrence of P. ulmi infesting grape vines was reported in Pirapora, state of Minas Gerais.

Panonychus ulmi was first reported in Brazil by Flechtmann (1967a) who discovered them on apples imported from Argentina. Soon after, Bleicher (1974) reported their presence on apple trees (Malus domestica L.) growing in orchards in the south of the country. However, European countries such as France, Austria, Portugal, Italy, and Greece consider the P. ulmi a grape vine pest [START_REF] Rambier | Les tétranyques nuisibles à la vigne en France continentale[END_REF][START_REF] Artofer | Spider-mites in vine cultivation[END_REF][START_REF] Carmona | O complexo Acarina nas culturas portuguesas[END_REF][START_REF] Girolami | Statistical and mathematical methods in population dynamics and pest control. Proceedings of meeting of the Meyer MKPS. 1974. A revision of the Tetranychidae of Africa (Acari) with a key to the genera of the world. Department of Agricultural Technical Services, Republic of South Africa[END_REF]Papaioannou-Souliotis et al. 1994). Similar reports were also registered in Morocco and the United States [START_REF] Reeves | Tetranychidae infesting woody plants in New York State, and a life history study of the elm mite Eotetranychus matthyssei n. sp[END_REF][START_REF] Tixier | Phytoseiid and tetranychid mites (Acari: Mesostigmata, Prostigmata) of some Moroccan crops[END_REF].

Despite reports of P. ulmi on apple trees as early as 1967 (Flechtmann, 1976a), grape vines in Brazil were not affected, in contrast to the damages the P.

ulmi caused to vineyards in Europe. [START_REF] Navia | Avaliação do risco de introdução de ácaros fitófagos associados à cultura da uva no Brasil[END_REF] hypothesized that the mites that attacked grape vines in Europe had different biotypes than those that infested apple trees. [START_REF] Moraes | Manual de acarologia -Acarologia básica e ácaros de plantas cultivadas no Brasil[END_REF] 

Introduction

With the increasing number of sequences being deposited in public DNA sequence databases, concerns about the taxonomic misidentification of the specimens used to obtain these sequences is also growing, which threats the utility of databases themselves. Several studies have stressed the problem of wrongly attributed sequences in databases [START_REF] Sperling | A DNA-based approach to the identification of insect species used for postmortem interval estimation[END_REF][START_REF] Ruedas | The importance of being earnest: what, if anything, constitutes a "specimen examined?[END_REF][START_REF] Vilgalys | Taxonomic misidentification in public DNA databases[END_REF], which in some cases is extreme, as in the study by [START_REF] Bridge | On the unreliability of published DNA sequences[END_REF] which revealed that more than 20% of the deposited sequences in public databases for three randomly chosen well-studied groups of fungi may be erroneous. While a potential source of error is poor DNA isolation techniques resulting in the amplification of incorrect or chimeric DNA sequences [START_REF] Bridge | On the unreliability of published DNA sequences[END_REF][START_REF] Hugenholtz | Chimeric 16S rDNA sequences of diverse origin are accumulating in the public databases[END_REF], or even disagreement among taxonomists in the use of plesiomorphic or homoplastic characters in the delimitation of species based on morphology [START_REF] Vilgalys | Taxonomic misidentification in public DNA databases[END_REF][START_REF] Diéguez-Uribeondo | Re-evaluation of the enigmatic species complex Saprolegnia diclina -Saprolegnia parasitica based on morphological, physiological and molecular data[END_REF], purely sample misidentification is a common important source of equivocal DNA sequences.

The problem of taxonomic inaccuracy and insufficient annotations in public DNA accessions threats the reliability of phylogenetic and ecological conclusions inferred from such sequences ( [START_REF] Holst-Jensen | On reliability[END_REF]), and are an obstacle for correct sequence-based species identification. They also reveal that the greatest challenge in the barcoding approach which is increasingly used to identify taxa (De Salle et al. 2005), will be of a taxonomical, rather than technical, nature [START_REF] Nilsson | Taxonomic Reliability of DNA Sequences in Public Sequence Databases: A Fungal Perspective[END_REF]. This difficulty has been cited not only for microorganisms as in Nilsson's paper, but examples on mammals, birds and arthropods are increasingly being reported. In mammalian and avian groups, [START_REF] Ruedas | The importance of being earnest: what, if anything, constitutes a "specimen examined?[END_REF] cited a number of molecular studies with suspected results. The subsequent examination of documented voucher specimens from which DNA was extracted and sequenced, revealed misidentification of some species or incomplete identification of others, in some cases now known to constitute complexes of sibling species. The problem of wrongly attributed data is becoming well-known in biological studies, and the need to deposit voucher material in public collections has been repeatedly advocated [START_REF] Hawksworth | Misidentification" in fungal DNA sequence databanks[END_REF]). In the work by [START_REF] Ruedas | The importance of being earnest: what, if anything, constitutes a "specimen examined?[END_REF] it was found that 73% (41 out of 56) of the papers surveyed in the last four numbers of the specialized journal Molecular and Phylogenetics and Evolution, did not link their sequence data to a voucher specimen at all, making it impossible to verify the validity of species identification. The well studied mosquitoes of the anophiline group which is characterized by the presence of species complexes that are difficult to differentiate using morphological criteria alone, is another relevant example of species misidentification. Reviewing 178 GenBank accession numbers matching the nuclear ribosomal ITS2 sequences of Latin American anophelines, Marrelli et al. (2006) showed that intraspecific variation ranged from 0.2% to 19%, concluding that misidentification and/or sequencing errors may be responsible for some of the high divergence values. The high number and diversity of examples of misidentification data in DNA sequence databases let suspect this type of error in other taxonomical groups not yet examined.

Among the phytophagous mites of the Tetranychidae family, several species are of high economic and quarantine importance in agriculture. The genus Tetranychus Dufour, 1832 gathers 142 valid species (Migeon and Dorkeld 2006) and includes many significant crop pests (Jeppson et al. 1975). In this family, the genus Tetranychus provides a good example of organisms where species identification might be misleading. Systematics has traditionally relied on a diversity of few minute morphological characters, mainly based on microscopic examination. As a result, morphological based identification of Tetranychus species requires advanced taxonomic expertise, especially in morphologically very close taxa, as the species belonging to the group Tetranychus (Tetranychus), also referred as Tetranychus sensu stricto, from the ninth species-group which was defined by Flechtmann and Knihinicki (2002). In this group, the two spotted spider mite, Tetranychus urticae Koch, 1836, a highly polyphagous species causing outbreaks in many crops worldwide, is certainly the most studied species. Two forms of this species, distinguished by their body color, either green or red, have been recognized. The color of the mite body results from different haemolymph pigments [START_REF] De Boer | Reproductive barriers[END_REF]. However, the red form, also called T. cinnabarinus (Boisduval 1867), has been regarded in some cases as a distinct species (Boudreaux 1956;Boudreaux andDosse 1963a, b, Meyer, 1974) and its taxonomical status is a long standing issue continuously addressed in the literature. The most accepted taxonomical position does not recognize T.

cinnabarinus as a valid species (e.g. Pritchard and Baker 1955;Dupont 1979;Meyer 1987;Baker and Tuttle 1994) as in the World Catalogue of Spider Mite (Bolland et al. 1998), which is rather cited as a synonym of T. urticae. This taxonomical controversy, although newly addressed by molecular approaches (Navajas et al. 1998), remains yet unsolved. While some studies concluded that the two forms are synonymous (Gotoh and Tokioka 1996;Ehara 1999;Hinomoto et al. 2001) and others do not achieve to separate them [START_REF] Xie | Genetic relationship between the carmine spider mite Tetranychus cinnabarinus (Boisduval) and the two-spotted mite T. urticae Koch in China based on the mtDNA and rDNA ITS2 sequences[END_REF], other recent work using DNA sequences concluded on to the validity of the species T.

cinnabarinus (Li et al. 2009), reviving its controversial taxonomic status.

The large range of geographical and ecological diversity of the two spotted spider mite and its economic importance, has motivated many studies on the genetic diversity of this species (Navajas et al. 1998;Tsagkarakou et al. 1999;Navajas et al. 2000;Hinomoto et al. 2001;[START_REF] Takafuji | Host-related differences in diapause characteristics of different geographical populations of the Kanzawa spider mite, Tetranychus kanzawai Kishida (Acari: Tetranychidae), in Japan[END_REF]Navajas et al. 2002;Osakabe et al. 2002;[START_REF] Xie | Population structure of the two-spotted spider mite (Acari:Tetranychidae) from China[END_REF]Hinomoto et al. 2007;[START_REF] Ros | Spider mite (Acari: Tetranychidae) mitochondrial COI phylogeny reviewed: host plant relationships, phylogeography, reproductive parasites and barcoding[END_REF][START_REF] Xie | Genetic relationship between the carmine spider mite Tetranychus cinnabarinus (Boisduval) and the two-spotted mite T. urticae Koch in China based on the mtDNA and rDNA ITS2 sequences[END_REF]Li et al. 2009) (2007) firstly reported on this issue, giving an example of thirteen COI sequences deposited in Genbank as T. urticae which likely were T. truncatus [START_REF] Ehara | Notes on some tetranychid mites of Japan[END_REF] In some cases, while the deposited sequences are linked to a publication, the methods used for species identification are not clearly mentioned e. g. [START_REF] Xie | Population structure of the two-spotted spider mite (Acari:Tetranychidae) from China[END_REF], [START_REF] Xie | Genetic relationship between the carmine spider mite Tetranychus cinnabarinus (Boisduval) and the two-spotted mite T. urticae Koch in China based on the mtDNA and rDNA ITS2 sequences[END_REF] and Li et al. (2009) which adds ambiguity to species identification. Likewise, more than 50% of the Tetranychus sequences found in GenBank have been directly submitted without having been recorded in published documents [START_REF] Ros | Spider mite (Acari: Tetranychidae) mitochondrial COI phylogeny reviewed: host plant relationships, phylogeography, reproductive parasites and barcoding[END_REF]. All these data raise the question of the reliability of DNA sequences of T. urticae and close related species that could result in misleading taxonomic identifications or equivocal phylogenetic inferences.

In this study, we evaluate the identity of DNA sequences available in Genbank which were deposited as T. urticae, together with those of some closely related taxa, mainly red pigmented species, which are important crop pests that might easily be misidentified among spider mites. Sequences of both ITS and COI available in Genbank were retrieved. In addition sequences of eighteen new T.

urticae samples unambiguously identified by morphological criteria were generated in this study and included in the analyzed dataset. This study uncovered that several DNA sequences publically available having been attributed to economically relevant taxa, are incorrectly named. In particularly it sheds new light in the controversial taxonomic status of T. cinnabarinus and highlights the need of using combined morphological and molecular approaches to guaranty reliability of accessions in public databases.

Material and methods

Field samples

A total of 18 samples of T. urticae were collected from several host plants in 15 different localities: Brazil (1), France [START_REF]Among the pairs sampled, approximately 20% indicated a high level of distance (≥ 6%)[END_REF], Greece (1) and Spain (10) (Table 1).

Most of the samples included the red form of T. urticae, except mites from Greece and from Brazil which were all green. At least 50 individual mites were collected in each locality. Mites were preserved in 100% ethanol for molecular analysis and in 70% ethanol for morphological identification.

Mite species identification

To ensure the best reliability between the species identification and their attributed DNA sequences, a combined approach based on morphological and molecular data was undertaken.

Morphological-based identification: Specimens were directly mounted on microscopic preparations in Hoyers' medium, and discriminative characters were examined under phase contrast and interference microscopes in 40x and 100x objectives (Leitz Wetzlar and Nikon Eclipse 80i). From each population, 25 females (dorso-ventral position) and ten males (lateral position) were mounted to identify the species by morphological analysis. When only a few males were present in a sample, (which is usual for Tetranychus species because of femalebiased sex-ratio), all of them were slide-mounted. Morphological identification was performed by examining relevant taxonomic characters for Tetranychidae systematics. As it is the rule in the genus Tetranychus, the shape of the aedeagus (male genitalia) was used to discriminate the species using the pertinent literature (Pritchard and Baker 1955;Meyer 1974Meyer , 1987;;Baker and Tuttle 1994;Ehara, 1999) and these were compared with specimens deposited at the reference collection of the Unidade de Acarologia, Laboratório de Quarentena Vegetal, Embrapa Recursos Genéticos e Biotecnologia (Cenargen), Embrapa Cenargen, Brasilia, Brazil. The studied material was also deposited in this collection to serve as voucher specimens.

Molecular-based identification: Sequences of the COI and ITS were also obtained for specimens of all the 18 samples in Table 1, which were species identified based on morphological reliable criteria. One to three individual PCR products per population were sequenced. These sequences have been used as reference for the two forms of T. urticae (green and red) in a phylogenetic analysis.

DNA extraction, PCR amplification and sequencing

Genomic DNA was extracted from single adult females using the DNeasy tissue Kit (Qiagen, USA), following the protocol for animal cultured cells. Manufacturer's instructions were modified for DNA extraction of small mites, as follows. After removing all of the ethanol used to preserve the mites, 90 µl of phosphate-buffered saline (PBS) buffer was added to a 1.5 ml microcentrifuge tube. Mites were not crushed. In the next step, 10µl of proteinase K 20 mg ml -1 (40mAU mg -1 protein)

was added followed by 100 µl of buffer AL provided in the Qiagen Kit. Tubes were incubated at 70ºC for 1-2 h. The following steps of the standard Qiagen protocol were applied, except that volumes were reduced by half. DNA was recovered in a 1.5 ml microcentrifuge tube adding 50 µl of ultrapure water on the membrane of the mini spin column.

Two target DNA fragments were PCR-amplified and sequenced per single mite: the nuclear ribosomal region spanning the ITS1, 5.8S and ITS2 region (about 1200 bp) and a fragment of the mitochondrial COI gene (about 950 bp). The amplification reactions were carried out in 25 µL volumes containing 2.5 µl of 10x buffer supplied by the manufacturer, 0.13 un/µL (5 units) of Taq polymerase (Qiagen), 2 µL DNTP (0.25 mM of each base), 0.5 µM of each oligonucleotide primer (10 mM), 2 µl of MgCl 2 (25 mM), 15.37 µL or 13.37 µL water and 2 or 4 µL of the template DNA for ITS and COI, respectively. Samples were denatured at Sequence and phylogenetic analyses Sequences were aligned using the CLUSTAL W Multiple alignment procedure (Thompson et al. 1994) implemented in the BIOEDIT software, version 7.0.4 (Hall 1999). No manual adjustments to the CLUSTAL alignment were performed.

Estimates of nucleotide polymorphism, number of haplotypes and haplotype diversity were determined using DnaSP software version 5 [START_REF] Librado | DnaSP v5: A software for comprehensive analysis of DNA polymorphism data[END_REF]. Pairwise nucleotide distances were calculated using Kimura's twoparameter correction and the software MEGA 4 (Kimura 1980;Tamura et al. 2007) for ITS2 and COI sequences of tetranychid species considered in this work.

Phylogenetic relationships were estimated based on both ITS2 and COI sequence alignments using the maximum likelihood (ML) method implemented in PhyML version 3 (Guidon and Gascuel 2003). ModelTest version 3.8 [START_REF] Posada | Modeltest: testing the model of DNA substitution[END_REF]) was used to estimate the substitution model by hierarchical likelihood ratio test (hLRT) and approximated Akaike information criterion (AIC).

The ModelTest server [START_REF] Posada | ModelTest Server: a web-based tool for the statistical selection of models of nucleotide substitution online[END_REF]), a web-based application, was used to execute the Modeltest computation. The likelihood scores for 56 models of DNA substitution was performed using PAUP*4.0 beta 5 software (Phylogenetic Analysis Using Parsimony) [START_REF] Swofford | PAUP* Phylogenetic Analysis Using Parsimony (*and Other Methods) Version[END_REF]. The best-fit model, HKY85, [START_REF] Hasegawa | Dating of the human-ape splitting by a molecular clock of mitochondrial DNA[END_REF] which takes into account a variable transition and transversion frequencies (Ti/Tv ratio =1.4084), was used for phylogenetic analysis of the ITS2 sequence dataset. In the case of COI sequences, the general times reversible GTR (GTR + I + G) model [START_REF] Lanave | A new method for calculating evolutionary substitution rates[END_REF]Tavare 1986;[START_REF] Rodriguez | The general stochastic model of nucleotide substitution[END_REF]) which takes into account a proportion of invariable sites (0.6285) and a gamma distribution for substitution rate heterogeneity (2.1037) was selected.

Robustness was evaluated with 1000 bootstraps replicates, and the approximate likelihood ratio test (aLRT) function within PhyML [START_REF] Mendonça | Approximate likelihood ratio test for branchs: A fast, accurate and powerful alternative[END_REF] was used to test the accuracy of each branch using the log-likelihood test. 1996) and can help reconstruct historical and biogeographical scenarios (Navajas et al. 1998;Navia et al. 2005).

The most used markers in molecular evolution include the Internal Transcribed Spacer (ITS) region of ribosomal DNA (rDNA) and a fragment of mitochondrial DNA (mtDNA), the Citocromo Oxidase subunit I (COI) gene.

Mitochondrial DNA has maternal inheritance, a haploid mode of transmission throughout the generations where there are no re-combinations and the genes are a unique genetic loco [START_REF] Avise | Intraspecific phylogeography: the mitochondrial DNA bridge between population genetics and systematics[END_REF][START_REF] Hoy | Insect molecular genetics: an introduction to principles and applications[END_REF]Roderick 1996).

Phylogenies inferred from mitochondrial DNA together with the geographic information on the populations provide ways to examine the geographic structure of these populations [START_REF] Avise | Molecular Markers, Natural History and Evolution[END_REF]Brower et al. 1996;Hillis et al. 1996). These analyses allow access the patterns of phylogeographic distribution of the studied species (Roderick 1996;[START_REF] Avise | Phylogeograph. The history and formation of species[END_REF][START_REF] Navia | Ácaros Eriophyoidea (Prostigmata) associados a palmeiras (Arecaceae), com ênfase no ácaro do coqueiro, Aceria guerreronis Keiferespectro de hospedeiros e aspectos biogeográficos. Tese (Doutorado)[END_REF], and this information is important to exploring colonization routes and the migratory history of the populations. It can additionally be used to suggest or trace the origin of the species, in cases of accidentally introducing exotic pests into new regions (Navajas 1998;[START_REF] Baliraine | Population genetics of the potencially invasive African fruit fly species, Ceratitis rosa and Ceratitis fasciventris (Diptera: Te´hritidae)[END_REF]Navia et al. 2005;[START_REF] Lozier | Evolutionarily significant units in natural enemies: Identifying regional populations of Aphidius transcaspicus (Hymenoptera: Braconidae) for use in biological control of mealy plum aphid[END_REF]). The COI fragment can be useful in intraspecific and interspecific analyses of phylogenetic relationships among populations of species of the genus Tetranychus (Navajas et al. 1996(Navajas et al. , 1996a;;1998;Navajas 1998;Hinomoto et al. 2001;[START_REF] Breeuwer | most likely due to the inclusion of taxa in the final data set compiled by the authors, which do not correspond to T. urticae. The phylogenetic tree (Figure 3) indicated the presence of different groupings among the populations examined and two major clades highly supported by bootstrap values were observed: Clade 1, composed of populations from Japan (17)[END_REF][START_REF] Nishimura | The genetic variation among populations of Tetranychus kanzaway complex (Acari: Tetranychidade) indicating by using mitochondrial, ribosomal, and microsatellite DNA markers[END_REF]).

The ITS region in the nuclear genome is diploid and recombination might occur. Sequences from the ITS2 region have been widely used to analyze phylogenetic relationships, to study intra and interspecific variation and for species diagnostics. For example, in eriophyde mites, the differences observed between the sequences of the ITS1 region were used to identify species of the genus Cecidophyopsis (Acari: Eriophyidae) [START_REF] Kumar | Identification of Cecidophyopsis mites (Acari : Eriophyidae) based on variable simple sequence repeats of ribosomal DNA internal transcribed spacer-1 sequences via multiplex PCR[END_REF]. In insects, the intra specific variability and the definition of complexes in populations of the mosquito Anopheles, were defined based on the analysis of TS2 sequences [START_REF] Fairley | Intragenomic heterogeneity of internal transcribed spacer rDNA in neotropical malaria vector Anopheles aquasalis (Diptera : Culicidae)[END_REF]Marrelli et al. 2006). Navajas (2001) and Navajas et al. (2002) reported that the ITS2 region (rDNA) together with morphological studies and crossbreeding is very useful for distinguishing closely related taxa in mites (Acari: Tetranychidae).

The authors used ITS2 sequences not only to distinguish between Tetranychus urticae and T. turkestani Ugarov & Nikolskii species, but also to establish a synonymy between T. kanzawai Kishida and T. hydrangea Pritchard & Baker.

Some studies have been conducted to examine the diversity and genetic structure of T. urticae, explore the factors that affect these variations, as well as infer the origin of its populations [START_REF] Tsagkarakou | Population structure in the spider mite Tetranychus urticae on Crete based on multiple allozymes[END_REF]1998;Navajas 1998;Navajas et al. 1998;Navajas & Boursot 2003;Hinomoto et al. 2001;[START_REF] Breeuwer | most likely due to the inclusion of taxa in the final data set compiled by the authors, which do not correspond to T. urticae. The phylogenetic tree (Figure 3) indicated the presence of different groupings among the populations examined and two major clades highly supported by bootstrap values were observed: Clade 1, composed of populations from Japan (17)[END_REF][START_REF] Breeuwer | most likely due to the inclusion of taxa in the final data set compiled by the authors, which do not correspond to T. urticae. The phylogenetic tree (Figure 3) indicated the presence of different groupings among the populations examined and two major clades highly supported by bootstrap values were observed: Clade 1, composed of populations from Japan (17)[END_REF]. Some results obtained are discordant and inconclusive. A 

Field samples

A total of 59 samples of T. urticae were collected from several host plants in nine different countries. Twenty-two populations were collected in Brazil (Figure 1, Table 1). The other populations were from France (15), Insular Spain (10), Canary Islands (5), Greece (1), Norway (1), Poland (1) Syria (2), Tunisia (1) and Canada

(1) ( 

Morphological mite species identification

Specimens were directly mounted on microscopic slide preparations in Hoyers' medium, and discriminative characters were examined under phase contrast and interference microscopes in 40x and 100x objectives (Leitz Wetzlar and Nikon Eclipse 80i). From each population, 25 females (dorso-ventral position)

and ten males (lateral position) were mounted to identify the species by morphological analysis. When only a few males were present in a sample, (which is usual for Tetranychus species because of female-biased sex-ratio), all of them were slide-mounted. Morphological identification was performed by examining relevant taxonomic characters for Tetranychidae systematics. As it is the rule in the genus Tetranychus, the shape of the aedeagus (male genitalia) was used to discriminate the species using the pertinent literature (Pritchard & Baker 1955, Meyer 1974, 1987, Baker & Tuttle 1994, Ehara, 1999) and these were compared with specimens deposited at the reference collection of the Unidade de Acarologia, Laboratório de Quarentena Vegetal, Embrapa Recursos Genéticos e Biotecnologia (Cenargen), Embrapa Cenargen, Brasilia, Brazil. The studied material was also deposited in this collection to serve as voucher specimens.

DNA extraction, PCR amplification and sequencing

Sequences of the COI and ITS were obtained for specimens of the samples in Table 1 and2, which were species identified based on morphological criteria.

One to three individual PCR products per population were sequenced. Genomic DNA was extracted from single adult females using the DNeasy tissue Kit (Qiagen, USA), following the protocol for animal cultured cells. Manufacturer's instructions were modified for DNA extraction of small mites, as follows. After removing all of the ethanol used to preserve the mites, 90 µl of phosphate-buffered saline (PBS)

buffer was added to a 1.5 ml microcentrifuge tube. Mites were not crushed. In the next step, 10µl of proteinase K 20 mg ml -1 (40mAU mg -1 protein) was added followed by 100 µl of buffer AL provided in the Qiagen Kit. Tubes were incubated at 70ºC for 1-2 h. The following steps of the standard Qiagen protocol were applied, except that volumes were reduced by half. DNA was recovered in a 1.5 ml The genetic differentiation between populations was also estimate using a modified Exact Test of Samples Differentiation based upon haplotype frequencies [START_REF] Raymond | An exact test for population differentiation[END_REF] with 100,000 steps in Markov chain as implemented in ARLEQUIN [START_REF] Excoffier | Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction data[END_REF][START_REF] Excoffier | Arlequin ver. 3.0: An integrated software package for population genetics data analysis[END_REF].

RESULTS AND DISCUSSION

Fifty-five new ITS (rDNA) sequences were obtained from T. urticae collected from different species of host plants in 8 countries, including 22 populations found in Brazil (Tables 1 and2). The efficiency of the protocol and the respective primers in amplification was 80%. urticae sequences obtained in this study revealed the same lengh variation recorded in the literature on other tetranychids (Navajas et al. 1999;Osakabe et al. 2002;[START_REF] Ben-David | ITS2 sequences as barcodes for identifying and analyzing spider mites (Acari: Tetranychidae)[END_REF]).

The ITS1 and ITS2 fragments were examined in order to find out if these regions, taken separately, were useful for studies on variability and genetic structure of T. urticae populations. For the ITS1 region, 59 sequences were analyzed (55 from this study and 4 from the GenBank), with a final length in the dataset of about 517 base pairs. For the ITS2 region, 82 sequences (55 new ones and 27 from GenBank) with about 488 bp were analyzed. Most of the ITS region sequences deposited in the GenBank (Suppementary Material, Appendix 1) for T.

urticae come from the ITS2 region. Alignments of sequences implied only very short insertion/deletion and did not pose any problems.

An intraspecific comparison was estimated for the ITS1 and ITS2 regions (Table 4). The number of polymorphic sites, the average number of differences in nucleotides (K), and the diversity of nucleotides (Pi) showed that the variability found in the ITS1 region was greater than in the ITS2 region. The diversity values of nucleotides were low and varied between 0.07% (ITS2) and 0.63% (ITS1). Navajas & Boursot (2003) found reduced variation in the ITS2 region (Pi < 0.5%) in their analysis of T. urticae sequences. The genetic distance of the base pairs was calculated for the ITS1 and ITS2 fragments K2P). The variation between the distances and the relative frequency of the pairs differed for both fragments in T. urticae sequences. The values observed varied between 0% and 1.7% for ITS1

and were lower for the ITS2 region, varying between 0% and 0.9%.

Trees were built using Neighbor-Joining (NJ) (K2P), considering the ITS1 and ITS2 fragments separately (not present here). There was no evidence of Navajas & Boursot (2003), [START_REF] Ben-David | ITS2 sequences as barcodes for identifying and analyzing spider mites (Acari: Tetranychidae)[END_REF] and [START_REF] Hurtado | Sequence analysis of the ribosomal internal transcribed spacers region in spider mites (Prostigmata: Tetranychidae) occurring in citrus orchards in Eastern Spain: use for species discrimination[END_REF]. The results indicated an absence of variability in the ITS2 region in 86% of the T. urticae sequences analyzed. Low levels of intraspecific divergence in the ITS2 region allow small differences in this region to be useful for interspecific distinction [START_REF] Hurtado | Sequence analysis of the ribosomal internal transcribed spacers region in spider mites (Prostigmata: Tetranychidae) occurring in citrus orchards in Eastern Spain: use for species discrimination[END_REF], which confirms its usefulness in diagnosis (Navajas et al. 1997;2001;[START_REF] Cruickshank | Molecular markers for the phylogenetics of mites and ticks[END_REF]Navajas 2001).

The COI region of mitochondrial DNA Size of the COI sequences obtained ranged from 800 to 955 bp and was greater in size than normally recorded in literature on COI sequences of T. urticae and other tetranychids [START_REF] Fournier | Mitochondrial DNA from a spider mite: isolation, restriction map and partial sequence of the cytochrome oxidase subunit I gene[END_REF]Navajas et al. 1996;Hinomoto et al. 2001;Hinomoto & Takafuji 2001;Hinomoto & Tanafuji 2004). Different primer combinations were used to generate COI GenBank sequences in the various studies, producing dissimilarities in size and the gene portion sequenced. A central fragment of 390 bp was present in the majority of accessions and was selected for subsequent analysis based on 57 sequences (20 new and 37 taken from the GenBank). Alignment was obtained without ambiguity and no insertion or deletion was observed. The results of the descriptive analyses for COI sequences have been summarized in Table 5. The COI region showed greater variability than ITS regions. The number of polymorphic sites, the average number of differences in nucleotides (K), and the diversity of nucleotides (Pi) were higher even when the smaller sample group consisting only to the new sequences generated in this study was examined. The Pi value for the combination of all the sequences was 3.25%. The extensive diversity of nucleotides in the COI region of the mitochondrial DNA (3% to 4%) compared to the reduced variation found in the ITS2 region of the ribosomal DNA (<0.5%), was described previously by Navajas & Boursot (2003). Twenty-one haplotypes were identified among the analyzed T. urticae populations, and a high haplotypic diversity (Hd = 0,8935) was noted.

The results of molecular distance matrix (K2P distance model, Kimura 1980) indicated considerable variation in genetic distances among the populations studied. Intraspecific diversity was high and reached the maximum value of 0.063

According to the phylogenetic tree (Figure 3), T. urticae populations did not group together according to the host plant where they collected, which is consistent with previous studies (Navajas 1998, Navajas et al. 1998;[START_REF] Breeuwer | most likely due to the inclusion of taxa in the final data set compiled by the authors, which do not correspond to T. urticae. The phylogenetic tree (Figure 3) indicated the presence of different groupings among the populations examined and two major clades highly supported by bootstrap values were observed: Clade 1, composed of populations from Japan (17)[END_REF]). In the groupings observed within the two main clades, the influence of the host plant effect was not perceived either (Figure 3 and Table 6).

Haplotype 1, which is highlighted in light blue on the phylogenetic tree, was represented by 13 T. urticae sequences obtained from specimens collected from eight different plant species. It happened also in Haplotypes 2 and 10 represented in the same way on the phylogenetic tree (Figure 3). A recent study based on COI and ITS2 regions as well as microsatellites (rDNA) was undertaken in Japan on the complex Tetranychus kanzawai Kishida 1927, a close species to T. urticae. In this case, results showed that the observed groups of populations emerged due to the effect of the host plant, rather to a geographic effect [START_REF] Nishimura | The genetic variation among populations of Tetranychus kanzaway complex (Acari: Tetranychidade) indicating by using mitochondrial, ribosomal, and microsatellite DNA markers[END_REF].

AMOVA results -Genetic structure of Tetranychus urticae populations related to geographic origin and host plants based on COI mitochondrial sequences

Analysis of Molecular Variance (AMOVA) was performed to investigate the geographical effect in the structure of T. urticae populations, as well as the host plant where the species was collected.

AMOVA results (Table 7) were significant with genetic variation of 60.76% spread across the planet, followed by rice, bananas, yam, sugarcane, citrus fruits, many fruit-bearing seedlings from East Asia and India, cereals from Europe, among others. This exchange of plants expanded the choice of foods being offered, and to this day it plays a strong role in disseminating pests. Therefore, haplotypes 1 and 10 could have been introduced in Brazil as a result of trade along historical routes during the age of exploration.

The greatest diversity of haplotypes was found among populations collected in countries bordering the Mediterranean Sea (15 haplotypes), followed by Asia (6 haplotypes). Of the Asia haplotypes, four were not found among the populations sampled in the Palearctic region (1, 2, 7 and 19). The haplotypes 3 (in Holland and Japan) and 5 (in Spain and Japan) were the only two haplotypes found in Asia and the Palearctic region. Haplotype sharing was also noted between Italy and Greece (haplotype 9), and between France and Spain (haplotype 17 region).

The results indicated a greater diversity of haplotypes among countries bordering the Mediterranean Sea and they were consistent according to ITS2 and COI regions, which supports the hypothesis that this region is where T. urticae species originated and from where it developed subgroups and colonized other regions of the world (Navajas 1998).

It is still not possible to present a complete scenario on the genetic diversity of the two-spotted spider mite (T. urticae) on a global scale due to insufficient and/or lack of information on regions like North America, Central America, Oceania, the Central and Southern regions of Africa, and on countries stretching between the Palearctic region and East Asia. Furthermore, due to the heterogeneity of the number of populations gathered in certain countries where the two-spotted spider mite has already been sampled. 

Figure 1 .

 1 Figure 1. States and their respective sampling points for collecting Tetranychus urticae in Brazil between October 2004 and July 2008 …………………………………………….…………………...

Figure 2 .

 2 Figure 2. A. World map of Tetranychus urticae survey between 10/2004 and 07/2008. B. Detail of the studied sites in France and Spain ……………………………………………………………....

Figure

  Figure3. Phylogeny tree inferred from mtDNA COI sequences of Tetranychus urticae retrieved from Genbank and obtained in this study from T. urticae field samples. The colors highlight the haplotypes 1, 2 and 10.The haplotype number and Genbank accessions are given (see Table6). The Neighbor-Joining (NJ) method was used based on distances calculated using Kimura's two-parameter (K2P) correction. Bootstrap values (higher than 50%) are near to the branches. The scale bar indicates the number of changes per site in NJ(K2P) inference …………………….

Figure 4 .

 4 Figure 4. Distribution and frequency of Tetranychus urticae ITS2 haplotypes (ribosomal DNA) in the world. ITS sequences retrieved from Genbank and those obtained in this study were included ……………………………………………..……………………………………………………...

Figure 5 .

 5 Figure 5. Distribution and frequency of Tetranychus urticae COI haplotypes (mitochondrial DNA) in the world. COI sequences retrieved from Genbank and those obtained in this study were included ………………………………………….…………………………………………………...

  . S. de. ÉTUDES TAXONOMIQUES DES ACARIENS TETRANYCHIDAE AU BRÉSIL, EN PARTICULIER SUR LA PHYLOGENIE ET LA STRUCTURE GENETIQUE DES POPULATIONS DE L´ACARIEN JAUNE, TETRANYCHUS URTICAE KOCH, INFERÉES À PARTIR DES SEQUENCES D´ADN RIBOSOMIQUE ET MITOCHONDRIAL. 2009, 198 p. Thèse (Doctorat), Institut des Sciences Biologiques, Université de Brasília, DF.

  . Les marqueurs génétiques sélectionnés dans ce travail sont les plus utilisés dans les recherches portant sur la systématique, la phylogénie des Tétranychidae. Lors de l'étude phylogénétique portant sur T. urticae, des séquences de cette espèce obtenues durant l´étude ont été analysées et comparées à celles d´espèces proches appartenant au groupe Tetranychus sensu stricto Tuttle & Baker (1968) (y compris T. cinnabarinus son synonyme potentiel), disponibles dans la base de données Genbank. Des incohérences au niveau de l'assignation de certaines séquences à certains taxa ont été révélées. Ces analyses sont présentées, discutées et des procédés visant à vérifier une plus grande fiabilité des bases de nucléotides à usage public sont suggérés. Ce document de thèse est rédigé en anglais et il comporte deux parties: la première comprend l'introduction et la présentation du contexte de la thèse, la deuxième rassemble quatre chapitres correspondant à des articles publiés, soumis ou en préparation (un publié dans International Journal of Acarology 1 , un soumis à Journal of Insect Science 2 , un à Experimental and Applied Acarology 3 et un article qui est actuellement en cours de rédaction 4 ).

  . Genomic DNA was extracted from single adult females from each population. The genetic markers chosen in this study are the most commonly used for research on systematics, phylogeny and evolution, and are very useful for studying populations of Tetranychidae mites. Tetranychus urticae populations used in the molecular studies were those collected in Brazil, plus populations collected in seven Palearctic countries with the support of the acarology staff of the Center for Biology and Management of Populations (CBGP), Monferriersur-Lez, France, oher collaborators from Europe, and including one sample from Canada (specimens from the mite rearing reference of the Genoma T. urticae Project). The obtained DNA sequences were used to investigate genetic variability, phylogeny, phylogeographic patterns, and the association to host plants in T. urticae populations collected in several regions of the world. During the course of the phylogenetic studies of the two-spotted spider mite, the sequences uncovered in this study and those available at GenBank were analyzed together with closely related species belonging to the group Tetranychus sensu stricto Tuttle & Baker 1968 (including its possible synonym T. cinnabarinus), obtained from Genbank. Inconsistencies were detected, leading to doubts on the reliability of data provided by GenBank.

  a survey of Tetranychidae mites in Brazil on different host plants and in different regions of the country 2) Describe new taxa for Tetranychidae family 3) Contribute to the molecular systematics of the genus Tetranychus 4) Investigate the factors affecting the genetic variability of T. urticae populations in a global context through phylogenetic analyses based on sequences of the nuclear and mitochondrial genome, including new sequences obtained from samples collected in Brazil and the Palearctic region, and inspect the geographic and host plants effects upon the genetic structure of T. urticae populations.

  Abad, R., Pina, T., Castañera, P., &Urbaneja, A. (2006). Comparative life history traits of four most abundant phytoseiids in Spanish mandarines feeding on the two-spotted spider mites, Tetranychus urticae. In: Program Book and Abstract, 12 th International Congress of Acarology, Amsterdam, The Netherlands, Jan Bruin (Ed.), August21-26, 2006 (p.3).Allam, L., Hmimina, M. & Ouahbi, A. (1999). Influence of host plant changing on the development of Tetranychus urticas Koch (Acari, Tetranychidae): consequence of mite infestation in Citrus fields. Journal of AppliedEntomology, 123(10),597-601. Amorim, D. S. (1994). Elementos básicos de sistemática filogenética. São Paulo: Sociedade Brasileira de Entomologia (314p). Anaraki, P.B., Arbabi, M. & Ajbishe, R.S. (2007). Study on different eggplant cultivars for infestation to two spotted spider mite (Tetranychus urticae complex) in Varamin region. Seed and Plant, .23,(1),2. Ansaloni, T., Miret, J.-A. J. & Ruíz, M. H. (2006). A new tool for Tetranychidae species molecular discrimination for quarantine purposes: a nested PCR-based techinique. In: Program Book and Abstract, 12 th International Congress of Acarology, Amsterdam, The Netherlands, Jan Bruin (Ed.), August 21-26, 2006 (p.12). Tetranychidae) a diversos acaricidas em morangueiro (Fragaria sp.) nos municípios de Atibaia e Piedade, SP. Ecossistema, 19, 40-46. Solignac, M., Periquet, G., Anxolabehere, D. & Petit, C. (1995). Phylogénies et classifications. In M. Sollignac & coll Génétique et evolution: l'espèce, l'évolution moléculaire. Tome 2 (pp.301-355). Paris: Hermann. ( 367p) Souza Filho, M. F. de, Suplicy Filho, N., Sato, M. E. & Takematsu, A. P. (1994). Suscetibilidade do ácaro rajado proveniente de videiras de Pilar do Sul, SP, a diversos acaricidas. Pesquisa Agropecuária Brasileira, 29(8), 1187-1192. Sperling, F. A. H., Anderson, G. S. & Hickey, D. A. (1994) A DNA-based approach to the identification of insect species used for postmortem interval estimation. Journal of Forensic Sciences, 39, 418-427. Suplicy Filho, N., Souza Filho, M. F. DE, Takematsu, A. P. & Sato M. E. (1994). Resistência do ácaro rajado Tetranychus urticae (Koch) à acaricidas em roseiras, na região de Itapevi, SP. Anais da Sociedade Entomológica do Brasil, 23(1), 51-55. Tanaka, M. A. de S., Betti, J. A. & Passos, F. A. (2000). Manejo Integrado de pragas do morangueiros. v.5., 61p. São Paulo: Secretaria da Agricultura e Abastecimento. (Manual Técnico, Série Especial). Thompson, J. D., Higgins, D.G., & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through weighting positionspecific gap penalties and weight matrix. Nucleic Acids Research, 22:4673-4680. Tian, T., Grafton-Cardwell, E. E. & Granett, J. (1992). Resistance of Tetranychus urticae Koch (Acari: Tetranychidae) to cyhexatin and fenbutatin-oxide in California pears. Journal of Economic Entomology, 85, 2088-2095. Tixier, M.S., Kreiter, S., Ferragut F. & Cheval, B. (2006). The suspected synonymy of Kampimodromus hmiminai and Kampimodromus adrianae (Acari : Phytoseiidae): morphological and molecular investigations. Canadian Journal of Zoology 84 (8): 1216-1222. Toda, S., Osakabe, M. H. & Komazaki, S. (2000). Interspecific diversity of mitochondrial CO1 sequences in Japanese Panonychus species (Acari: Tetranychidae). Experimental and Applied Acarology, 24, 821-829. Tsagkarakou, A., Navajas, M. & Lagnel, J.(1997). Population structure in the spider mite Tetranychus urticae on Crete based on multiple allozymes. Heredity, 7, 84-92. XIE, L., XIE, R.-R., ZHANG, K.-J. & HONG, X.-Y. (2008). Genetic relationship between the carmine spider mite Tetranychus cinnabarinus (Boisduval) and the twospotted mite T. urticae Koch in China based on the mtDNA and rDNA ITS2 sequences. Zootaxa 1726, 18-32. Yaninek, J.S. & Moraes, G. J. (1991). A synopsis of classical biological control of mites in agriculture. In F. Dusabeck V. Bukva (Eds). Modern acaralogy. v.1 (pp.133-149). Prague: Academia, The Hague SPB Academic Publication. Chapter 1 Two new spider mites (Acari: Tetranychidae) from Brazil: a Monoceronychus McGregor (Bryobiinae) from fingergrass and an Oligonychus Berlese (Tetranychinae) from grape * * * * (2010) International Journal of Acarology, 36(6) (December issue) Article accepted for publication in International Journal of Acarology. The page range is not yet known Renata

  Leg chaetotaxy, from coxa to tarsus (solenidia in parentheses). No alternative setal count was observed.I.2-1-9-2-9(1)-9(1) + 2 duplex (6 proximal and 4 distal to duplex) II.2-0-5-4-4-7(1) + 1 duplex (2 proximal and 6 distal) Measurements (n=2). Body length349-374; width 167-195. Smaller than female and less robust. Anterior median projection not covering the gnathosoma, thumblike, apically flat and slightly emarginate, not extending beyond seta v2 of the lateral projections. The dorsal integument on the median portion of the body granulated in between the slender transverse striae. Caudal three pairs of hysterosomal setae (f1, f2 and h1) more lanceolate-serrate than in the female and with smaller variation in length. The two males studied exhibited differences between the length of the opisthosomal setae, however the length ratios between f1, f2 and h1 remained constant.Leg chaetotaxy, from coxa to tarsus (solenidia in parentheses). No alternative setal count was observed. Measurements (n=10). Body length275-390; width 185-221. Similar to female; dorsal integument on the median portion of the body with stronger striae than in female and completely smooth. The integument around seta e1

FIGURE 1 - 7 :

 17 FIGURE 1-7: Monoceronychus tchecensis n. sp. Female. 1. Dorsal view; 2-5. Legs I-IV, (from coxa to tarsus), in this sequence; 6. Palptarsus; 7. Prodorsal anterior projection.

FIGURE 8 -

 8 FIGURE 8-14. Monoceronychus tchecensis n.sp. 8. Protonymph. Dorsal view; 9-14. Female. 9. Peritreme; 10. Genitoanal region; 11-14. Tarsal appendages of legs I-IV, in this sequence.

FIGURE 15- 22 .

 22 FIGURE 15-22. Monoceronychus tchecensis n. sp. Male. 15. Dorsal view; 16-19. Legs I-IV (from coxa to tarsus), in this sequence; 20. Genital system detail; 21. Prodorsal anterior projection; 22. Aedeagus.

FIGURE 23 -

 23 FIGURE 23-29. Monoceronychus tchecensis n. sp. Micrographs. Female. 23. Dorsal view; 24. Genitoanal region; 25. Ocelli. Male. 26. Genitoanal region. 27-29. Prodorsal anterior projection. 27. Female; 28. Male; 29. Protonymph.

Family

  having the prodorsal anterior projections entirely covering the gnathosoma. However it differs from these species in the length of leg I which is longer than the body (about as long as the body in M. linki and shorter in M. bambusicola); peritreme ending in two-three chambers (ending in anastomosed chambers in M. linki and in M. bambusicola); h1 setae (fifth pair of dorsocentrals) are long and robust (flagellate in M. bambusicola). typical Oligonychus with dorsal hysterosomal setae longer than longitudinal distances between bases of consecutive pairs. Tarsus I with three tactile and one sensory setae proximal to duplex; and two tactile setae ventrally beyond the duplex. Tarsus III and IV each with ten tactile and one sensory setae. Aedeagus upturned, stem at an angle slightly larger than 90º in relation to axis of shaft. The distal enlargement with the axis at an acute angle to that of shaft, with dorsal margin slightly sinuate and the tip bending down; anterior margin of knob acutely angulate. Tibia I of the male with seven tactile and four sensory setae; tarsus I with 11 tactile and three sensory setae.FEMALE. Measurements (n=4). Bodylength 356 (348-377); width 247 (218-247). Color in life carmine red. Dorsum. Dorsal striation almost longitudinal on propodosoma and transverse on hysterosoma, except for the area between setae f1 and f2, where the striation pattern becomes slightly transverse and irregular. Lobes of striae rounded, wider than long and separated at the base. Peritreme slender, slightly curved and enlarged distally. Gnathosoma. Rostrum reaching the distal third of femur I. Stylophore enlarged, rounded anteriorly. Palp tarsus: palptarsal thumb bears terminal eupathidium (spinneret) 5 (4-5) long and 5 (4-5) in diameter; two lateral eupathidia each 6 (6-7) long; solenidia 4 (4) long. Setae. Dorsal body setae long and variable in size. Setae sc1, c1, d1, e1, f1, f2 and h1 longer than distance between the bases of each member of the pair and all dorsal hysterosomal setae longer than longitudinal distances between bases of consecutive pairs. Prodorsal setae slender and slightly barbed. Prodorsal setal length: v2 65 (62-65), sc1 99 (91-99), sc2 78 (73-78). Distances between prodorsal setal insertions v2-v2 73 (65-73), sc1-sc1 88 (75-88), sc2-sc2 195 (182-195). Hysterosomal setae long, slender and pubescent. Hysterosomal setal length: c1 83 (83-86), c2 81 (81-86), c3 81 (81-83), d1 83 (83-86), d2 83 (83-91), e1 75 (75-81), e2 88 (86-94), f1 70 (70-73), f2 65 (62-70), h3 52 (49-52). Distances between hysterosomal setal insertions: c1-c1 65 (65-68), c2-c2 130 (122-130), c3-c3 221 (203-226), d1-d1 78 (70-78), d2-d2 166 (156-166), e1-e1 52 (49-52), e2-e2 120 (117-127), f1-f1 36 (34-39), f2-f2 49 (49-52), h1-h1 23 (23-29). Legs. Tarsi I, II, III and IV with tactile and sensory setae. Tibia I showing tactile and sensory setae; tibia II, III and IV presenting only tactile setae. Tarsus I bears 3 tactile setae and one solenidion proximal to the duplex setae; two tactile setae on venter just beyond the first duplex seta. Empodia with 4 pairs of proximo-ventral hairs. Leg chaetotaxy, from coxa to tarsus (solenidia in parentheses). No alternative setal count was observed. Ventral striae smooth, without lobes, transverse to pg setae except for the longitudinal pattern immediately anterior to the genital flap; flap with transverse striae. MALE. Measurements (n=4). Smaller than female. Only one male in dorsoventral position, body length 325; width 195, including rostrum. The other three males were in lateral position. Palptarsus: terminal eupathidium (spinneret) 5 long and 6 in diameter; two lateral eupathidia 5 long; solenidion 4 long. Legs. Tarsus I bears three tactile setae and three solenidia well proximal to the duplex setae; a single tactile seta on venter just beyond first duplex. Tarsus II bears 4 tactile setae and one solenidion well proximal to the duplex. Empodia provided with three pairs of proximo-ventral hairs.Leg chaetotaxy, from coxa to tarsus (solenidia in parentheses)Aedeagus upturned, stem at an angle slightly larger than 90º in relation to axis of shaft. The distal enlargement with the axis at an acute angle to that of shaft, with dorsal margin slightly sinuate and the tip bending down; anterior margin of knob acutely angulate.TYPE MATERIALHolotype female. Brasil, Minas Gerais, Pirapora, latitude 17º21' S, longitude 44º56'W, altitude 489m; ex Vitis vinifera L., grape; 12. iv. 2006 and 8.viii.2008; D. Navia and R. S. Mendonça. 

FIGURE 33 -

 33 FIGURE 33-35. Oligonychus fileno n. sp. Female. 33. Dorsal view; 34. Genitoanal region; 35. Palptarsus.

FIGURE 36- 44 .

 44 FIGURE[36][37][38][39][40][41][42][43][44]. Oligonychus fileno n. sp. Female.[36][37][38][39]. Tibia and tarsus of legsI-IV, in 

FIGURE 56 -

 56 FIGURE 56-58. Oligonychus fileno n. sp. Micrographs. Male. 56. Aedeagus. Female. 57. Genitoanal region; 58. Peritreme.

Figure 1 .

 1 Figure 1. States and their respective sampling points for collecting tetranychid mites

  same leaf. This occurred on bean (T. urticae and E. banksi; T. urticae and Mononychellus planki; T. urticae, T. ludeni and T. desertorum; T. desertorum and M. planki; as well as T. ludeni and M. planki); on soybean (T. urticae and M. planki; T. urticae and T. desertorum; T. urticae, T. desertorum and M. planki; T. ludeni and M. planki and T. desertorum and M. planki); on cotton (T. urticae and M. planki; T. ludeni and M. planki and; T. ludeni and T. mexicanus); on okra (T. neocaledonicus and M. planki); and eggplants (T. urticae e M. tanajoa). Tetranychus mexicanus, P. ulmi, O.

  Koch, 1836 Tetranychus urticae Koch, 1836. (Koch 1836 2 apud Pritchard and Baker 1955). Typehost: Urtica sp. Type-locality: Regensburg, Germany. Asteraceae Ambrosia polystachya DC. (ragweed), Itaqui, Rio Grande do Sul, 29.IX.2007. Parthenium sp. (parthenium), Universidade Federal de Santa Maria, Rio Grande do Sul, 26.IX.2007. Paschoal (1970a) first confirmed the presence of T. urticae on A. trifida L. in the state of São Paulo. Ambrosia polystachya is a new host for T. urticae in Brazil and in the world. Zingiberaceae Alpinia purpurata K. Schum (alpinia), Faculdade da Terra de Brasília, Recanto das Emas, Distrito Federal, 28.XI.2005.

  , and therefore a relatively large number of DNA sequences of T. urticae together with other close Tetranychus species have been deposited in public-access databases. Molecular discrimination between T. urticae and related species, and their phylogenetic position in the Tetranychidae, has generally been based on a restricted number of genomic regions. The two variable internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA (rDNA) and the mitochondrial Cytochrome Oxidase I (COI) gene are the most widely utilized. Recent studies however, contested the identity of some nucleotide sequences deposited in public DNA database. Hinomoto et al.

  general phylogeographic pattern was decribed for T. urticae populations collected around the world.Navajas et al. (1998) observed the presence of two distinct groupings, one composed of Mediterranean populations and the other of populations of mixed origin in the world. The Mediterranean grouping showed high genetic diversity and this region was regarded as the centre of the origin of the species that later developed subgroups and colonized other regions of the Northern hemisphere and the world. Similar results were obtained byHinomoto et al. (2001). The phylogeographic pattern described byNavajas et al. (1998) was not confirmed in the studies conducted by[START_REF] Breeuwer | most likely due to the inclusion of taxa in the final data set compiled by the authors, which do not correspond to T. urticae. The phylogenetic tree (Figure 3) indicated the presence of different groupings among the populations examined and two major clades highly supported by bootstrap values were observed: Clade 1, composed of populations from Japan (17)[END_REF]. The presence of reproductive parasites such as Wolbachia and Cardinium has also reported as affecting phylogenetic patterns.Another factor that may hinder obtaining the discordant results regarding the genetic structure of T. urticae populations is the use of sequences from the GenBank that do not correspond to the taxon T. urticae, but rather to closely related species to the group Tetranychus sensu stricto(Tutle & Baker, 1968) or even to taxa of which the synonymy with T. urticae remains highly controversial, like T. cinnabarinus Boisduval (Mendonça et al. 2010 1 ). For instance, Hinomoto et al. (2007) identified 13 sequences of the COI (mtDNA) region that had been wrongly deposited in the GenBank as T. urticae, when they are actually related to the T. truncatus Ehara sequence. This study investigated genetic variability, phylogenetic relationships and the association to host plants in T. urticae populations collected in several regions of the world using COI and ITS2 sequences. New ITS2 (n=55) and COI (n=20) sequences of T. urticae from Brazil and the Palearctic region were obtained and included in the analyses together with the T. urticae sequences available from the GenBank. The deposited sequences, such as T. cinnabarinus and other sequences suspected of being wrongly identified were not considered in the analyses. The distribution of intraspecific genetic variability was examined using AMOVA. To examine the genetic variation of T. urticae populations by analyzing ITS and COI sequences.2.To investigate the effect of geographic variation and host plant on the genetic structure of T. urticae populations around the world, including on the new sequences obtained from samples collected for this study. 1 Renata S. de Mendonça, Denise Navia, Ivone R. Diniz, Philippe Auger, Maria Navajas (2010) A critical review on some closely related species of Tetranychus sensu stricto Tuttle & Baker (1968) (Prostigmata: Tetranychidae) in the public DNA sequences database. Submitted to Experimental and Applied Acarology 3. To analyze the distribution and frequency of genetic variation in T. urticae populations around the world MATERIAL AND METHODS Molecular analyses were conducted at the Center for Biology and Management of Populations (CBGP), advanced laboratory in a joint research unit INRA/IRD/CIRAD/AGRO.M, Monferrier-sur-Lez, France and at the Laboratory of Acarology, Plant Quarantine, Embrapa Genetic Resources and Biotechnology, Brasília, DF, Brazil.

Figure 1 .

 1 Figure 1. States and their respective sampling points for collecting Tetranychus urticae in Brazil between October 2004 and July 2008.

  Integrated Package for Population Genetic Data Analysis website). The genetic structure among T. urticae population was analyzed considering the molecular differences between individual haplotypes, in addition to differences in haplotype frequencies, resulting in estimates of Fst values(Weir & Cockerhan 1984). Significance levels of the genetic-variance components as well as Fst values were estimated by use of 10,000 permutations. The magnitude of genetic differentiation between groups was based on the evaluation of Fst values[START_REF] Wright | Variability among and within natural populations[END_REF]): the range 0.0-0.05 reflects low genetic differentiation; 0.05-0.15 moderate differentiation; 0.15-0.25 high genetic differentiation, and values > 0.25 reflect very high genetic differentiation. Different components affecting T. urticae populations were defined to perform the AMOVA. The hierarchy of the region was first used to separate T. urticae populations into groups: Set 1 (28 sequences) and Set 2 (25 sequences). Set 1 grouped populations from the countries bordering the Mediterranean Sea and we included in this set five populations collected in the Canary Islands (Spain). Set 2 included sequences from Asia. These two dataset were named Mediterranean and Asian regions respectively. In the second analysis, T. urticae populations were evaluated by sets of host plants family from Mediterranean (Set 1) and Asian (Set 2) regions separately in order to evaluate the host plant effect within each region. Finally, T. urticae populations from Asian and Mediterranean regions, Brazil and Netherlands were compared, all forming separate sets to verify the geographical effect in the population structuring.

  Twenty new COI (mtDNA) sequences were obtained. Only one individual from Brazil was sequenced. The efficiency of protocol extraction and the primers used to amplify the COI region was of 40%. Regarding the molecular techniques, in several cases mitochondrial fragment amplifycation failed. It would be useful to rework the DNA extraction protocol or to design new primers to increasing the number of generated COI sequences of T. urticae samples from BrazilThe ITS region of ribosomal DNAThe ITS fragments obtained ranged from 1010 to 1218 base pairs (bp). The ITS1 region from 484 to 508bp and the ITS2 region from 464 to 488bp. The 18S gene renaged from 32 to 40 bp and the 28S gene from 30 to 32 bp. Tetranychus.

  associations to host plants or to a geographic pattern in the structure of T. urticae populations apart from a modest grouping that indicated a tendency toward geographic structure among populations gathered in the Canary Islands. However, the bootstrap values were invariably low at below 50 %, which did not allow for further discussions on genetic structures of T. urticae populations. The 22 populations from Brazil were dispersed through the various branches, and vaguely grouped into sequences from Poland, Norway, France, Spain, and Taiwan. ITS2 sequences indicated strong similarities between the analyzed populations. In the set composed of 82 sequences, 71 were identical, including 22 new amplified sequences from samples collected in Brazil. Two other groups of sequences were noted: the first made up of sequences from Holland and Taiwan (X99881 and AY750700); and the second, of two sequences obtained during the course of this research from Spain and the Canary Islands (40-3 and 97-1). The homogeneity between ITS2 sequences of Tetranychus species was recorded by Navajas et al. (1998),

  between populations from the Mediterranean and Asian regions (Fst value = 0.6975, p = 0) suggesting a structuring pattern of T. urticae populations by geographical region. The AMOVA results for the host plant effect showed no significant differences of the genetic variation for both sets of plants compared separately in the Mediterranean (Fst = 0.1762, p = 0.1417) and Asian (Fst = 0.0834, p = 0.2541) regions, indicating the host plant factor did not interfere in the structuring of the studied T. urticae populations Table 7. Analysis of molecular variance (AMOVA) of Tetranychus urticae populations from several host plants from two distinct geographical regions, Mediterranean and Asian regions, based on COI sequences of the mtDNA.Sstatistical evaluations using AMOVA and the Exact Test of Sample Differentiation based on Haplotype Frequency (Raymond & Rousset 1995) were conducted to detect perceptible differentiation between T. urticae populations grouped by hosts. The analyses were carried out by hosts' family from the Mediterranean and Asian regions separately to reduce the interference of the region factor. For the Mediterranean region, the Exact Test of Sample Differentiation was significant (≤ 0.05) between haplotypes frenquencies of plants from the Solanaceae and Rutaceae families (p = 0.0476) showing a genetic structuring related to the host plant. Considering the extent geographic of the Mediterranean region accessed in this study, the results for plant families could be affected by the size of this area. To investigate if the genetic differentiation found among T. urticae populations from Solanaceae and Rutaceae in the Mediterranean region was due to the influence of geographical factor further analyses were performed for a smaller geographical region. A new set was established, where most of sample was collected: southern region of France (Solanaceae, Fabaceae, and Apocynaceae Urticaceae), Spain (Rutacea and Solanacea) and Italy (Convolvulacea and Rutaceae). The results for the AMOVA and the Exact Test of

Figure 4 .

 4 Figure 4. Distribution and frequency of Tetranychus urticae ITS2 sequences (ribosomal DNA) in the world. ITS sequences retrieved from Genbank and those obtained in this study were included.

2Figure 5 .

 5 Figure 5. Distribution and frequency of Tetranychus urticae COI haplotypes (mitochondrial DNA) in the world. COI sequences retrieved from Genbank and those obtained in this study were included.

  

  

  

  

Table 7 .

 7 Analysis of molecular variance (AMOVA) of Tetranychus urticae populations from several host plants from two distinct geographical regions, Mediterranean and Asian regions, based on COI sequences of the mtDNA…………………………………………………………………

Table 8 .

 8 Analysis of molecular variance (AMOVA) of sets from Tetranychus urticae populations collected on several host plants in Asian region………………………………………………………..

Table 9 .

 9 Exact test of Sample Differentiation based COI haplotypes frequencies of Tetranycus urticae from Asian and Mediterranean regions, the Netherlands and Brazil……………………....

  this paper two new spider mites from Brazil are described -a

	Monoceronychus McGregor collected from weeping fingergrass, Eustachys
	distichophylla (Lagasca & Segura) Nees (Poaceae), in the State of Rio Grande do

Sul; and a Oligonychus Berlese, collected from grapes, Vitis vinifera L. (Vitaceae), in the State of Minas Gerais. Also, Eotetranychus smithi

Pritchard & Baker, 1955 is 

newly recorded from South America.

Material and Methods

A legal permit for collecting zoological material for scientific purposes was required by the Brazilian Institute for the Environment and Renewable Natural Resources (IBAMA); we were issued permit number 231/2006 CGFAU/IBAMA. Mites were collected from weeping fingergrass and grape leaves by washing and sieving. Specimens were preserved in 70% ethanol and mounted on microscope slides in Hoyers' medium.

The Monoceronychus specimens were collected in Itaara, State of Rio Grande do Sul, which has a subtropical humid climate with uniformly distributed precipitation averaging 1,714.2 mm/annum

  September has low precipitation; at least one month has a lowest average total precipitation of 60 mm[START_REF] Tonietto | A multicriteria climatic classification system for graping-growing regions worldwilde[END_REF][START_REF] Tonietto | Caracterização macroclimática e potencial enológico de diferentes regiões com vocação vitícola de Minas Gerais[END_REF]). The annual average temperature is 23.8°C, the maximum a verage temperature is 31.7°C in January and the minimum is 12.2°C in June(Conce ição, 2005). a small median anterior cone-like projection; leg I longer than body; peritreme ending in two -three large chambers; h1 (fifth pair of dorsocentrals) heavily barbed, two and a half times longer than f1; sc2 and c3 marginal about twice as long as other dorsal setae except h1. Leg I longer than body. Trochanter III with a serrate dorsal seta; femur, genu and tibia II, III, IV of all legs with two types of setae: slender serrate dorsal and filiform. Coxa with only filiform setae. Tibia and tarsus with tactile and sensory setae.

		Oligonychus mites
	were collected from grape leaves during the dry season in April 2006 and in August FEMALE. Measurements (n=6). Body length 510 (463-541) including rostrum;
	2008. width 273 (234-273). Color in life yellowish. Dorsum. Integument of the
	Microscopic preparations of specimens were examined under phase contrast propodosomal and opisthosomal shields granulated forming a circular pattern visible
	and differential interference microscopes (Leitz Wetzlar and Nikon Eclipse 80i, only under oil magnification: between these regions, integument transversely striated.
	respectively). Micrographs were obtained using a Nikon DS-Fil digital camera with a Propodosoma with three anterior well developed projections, covering the rostrum,
	DS-L2 on the Nikon Eclipse 80i phase contrast and interference microscope. palpi and entire gnathosoma; anterior median projection enlarged, subtriangular and
	Selected taxonomic characters were prepared with Helicon Focus Software 4.10 apically rounded and with a small cone-shaped structure, 73 (68-73) long; lateral
	(2007) using a Helicon Filter 4.5 (2007). ArcSoft Panorama Maker software v. anterior projections 52 (49-55) long, each bearing the spatulate and serrate vertical
	4.2.0.59 was used to prepare the final images.
	73). Distances between hysterosomal setal insertions: c1-c1 57-65, c2-c2 161-179,
	Family TETRANYCHIDAE Donnadieu, 1875 c3-c3 213-255, d1-d1 57-60, d2-d2 151-161, e1-e1 31-39, e2-e2 117-138, f1-f1 68-
	Subfamily BRYOBIINAE Berlese, 1913 78, f2-f2 91-96, h1-h1 29-37.
	Legs.	Tribe HYSTRICHONYCHINI Pritchard & Baker, 1955
		Monoceronychus, McGregor, 1945
		Monoceronychus tchecensis Mendonça, Navia & Flechtmann n. sp.
		(Figs 1-32)

Pirapora municipality in Northern Minas Gerais State -was classified as tropical humid with a wet season from November to March. The winter season, June to Measurements are given in micrometers (µm). Female holotype measurements are followed by the range of measurements of the paratypes in parentheses.

Setae were measured from the center of the setal base to the tip of the seta; distances between setae were measured from center to center of their bases. Holotypes and paratypes were deposited in the Mite Reference Collection at the Laboratório de Quarentena Vegetal, Embrapa Recursos Genéticos e Biotecnologia, Brasília, DF, Brazil. Paratypes were also deposited in the Mite Collection at Departamento de Entomologia, Fitopatologia e Zoologia Agrícola of the Escola Superior de Agricultura "Luiz de Queiroz", ESALQ, University of São Paulo, Piracicaba, São Paulo, Brazil. Diagnosis. A typical Monoceronychus with prodorsal anterior projections entirely covering the gnathosoma; anterior median projection subtriangular and apically rounded with seta (v2). The anterior median projection extends beyond the tip of v2. Peritreme bent, arched inwards, distally ending in two-three chambers. Gnathosoma. Rostrum reaching the base of femur I. Stylophore anteriorly rounded, about as long as broad. Palp tarsus. Palptarsal thumb bears 6 setiform structures, two simple, 3 subequal eupathidia and one "solenidion" longer than eupathidia.

Setae. Dorsal setae variable in size and shape. Prodorsal setae v2 and sc1 lanceolate to spatulate and serrate; sc2, c3, h1 marginal and serrate; sc1,

c1-2, d1-2, e1-2, f1-2 (dorsocentral and dorsolateral) 

lanceolate to spatulate and serrate.

Prodorsal setae length: v2 17 (13-17), sc1 21 (21-23), sc2 51 (

[49][50][51][52]

. Distances between prodorsal setal insertions v2-v2

47-57, sc1-sc1 130-138, sc2-sc2 208-234. 

Hysterosomal setae length: c1 18 (

17-26), c2 21 (21-29), c3 34 (31-39), d1 16 (16- 18), d2 16 (16-17), e1 10 (10-13), e2 17 (16-17)

, f1 34 (34), f2 21 (18-21), h1 70 (65-

  : the closest species to Oligonychus fileno n. sp. are O. anonaePaschoal, 1970 and O. poutericola Feres & Flechtmann, 1986. However the aedeagus of the new species presents the dorsal margin slightly sinuate, longer posterior margin and the tip bending down (dorsal margin of the knob straight in O.

			Chapter 2			
	Notes on Brazilian spider mites (Acari: Tetranychidae) -new hosts and
	localities * * * *						
	Article submetted to Journal of Insect Science (Sptember 16 th , 2010)	
		Mendonça RS 1 , Navia D 2 , Diniz IR 3 , Flechtmann CHW 4	
	1. Universidade de Brasília, UNB, Instituto de Ciências Biológicas, Dep. Zoologia,
	Campus Universitário Darcy Ribeiro, ICC Sul, Sala AT-159, Asa Norte, 70910-900,
	Brasília-DF,	Brazil;	Université	Montpellier	II,	Montpellier,	France
	(renatasm@cenargen.embrapa.br)				
	2. Embrapa Recursos Genéticos e Biotecnologia, Laboratório de Quarentena
	Vegetal, Parque Estação Biológica, W5 Norte Final, Caixa Postal 02372, 70.770-900
	Brasília-DF, Brazil (navia@cenargen.embrapa.br)			
	3. Universidade de Brasília, UNB, Instituto de Ciência Biológicas, Dep. Zoologia,
	Campus Universitário Darcy Ribeiro, ICC Sul, Sala AT-116, Asa Norte, 70910-900,
	Brasília-DF, Brazil (irdiniz@unb.br)				
	4. CNPq researcher, Escola Superior de Agricultura "Luiz de Queiroz" (ESALQ),
	Universidade de São Paulo, Cx. Postal 09, 13.418-900, Piracicaba-SP, Brazil
	(chwflech@esalq.usp.br)					

anonae; sharply sinuous and smaller in O. poutericola). Tarsus I of the male of O. fileno n. sp. with 11 tactile setae and three sensory setae (eight tactile and six sensory in O. anonae and 12 tactile and three sensory in O. poutericola). Oligonychus fileno n. sp. females have the peritreme slender, slightly curved and enlarged distally (straight and ending in a bulb in O. anonae; and straight and ending in a kidney-shaped opening in O. poutericola).

New Record

Eotetranychus smithi Pritchard & Baker, 1955

A collection of spider mites by D. Navia and P. Drumond from roses, Rosa spp. (Rosaceae), from Rio Branco,

Acre, Brazil (9º58'18.49" South and 67º49'5.74" West)

, included female and male specimens of E. smithi, which is herein reported for the first time in South America.

Table 1 .

 1 Tetranychid mite species (Tetranychinae) collected in Brazil between 10/2004 and 07/2008.

	Specie	Localities	Hosts	Collections
	1. Eutetranychus banksi (McGregor, 1914)	10	10	13
	2. Eotetranychus tremae De Leon, 1957	1		1
	3. E. smithi Pritchard & Baker, 1955	1		1
	4. Monoceronychus n. sp. Mendonça, Navia & Flechtmann, 2010	1		1
	5. Mononychellus planki (McGregor, 1950)	18		26
	6. M. tanajoa (Bondar, 1938)	17		23
	7. Oligonychus anonae Paschoal, 1970	1		1
	8. O. Ilicis (McGregor, 1917)	1		1
	9. O. n. sp. Mendonça, Navia & Flechtmann, 2010	1		1
	10. O. mangiferus (Rahman & Sapra, 1940)	2		3
	11. O. aff. mangiferus	1		1
	12. Panonychus ulmi (Koch, 1836)	1		1
	13. Tetranychus abacae Baker & Pritchard, 1962	1		1
	14. T. bastosi Tuttle, Baker & Sales, 1977	4		5
	15. T. desertorum Banks, 1900	15	11	19
	16. T. evansi Baker & Pritchard, 1960	1		2
	17. T. gloveri Baker & Pritchard, 1962	1		1
	18. T. ludeni Zacher, 1913	15	10	20
	19. T. mexicanus (McGregor, 1950)	14	12	23
	20. T. neocaledonicus André, 1933	4		5
	21. T. ogmophallos Ferreira & Flechtmann,1997	1		1
	22. T. urticae Koch, 1836	55	34	101

:

  Larrea tridentata J. M. Coult, Phacelia crenulata Torr. ex S. Watson. Type-locality: Mesilla, United States.

	Fabaceae
	Mucuna aterrima Holland (black mucuna), Instituto Agronômico de Campinas,
	ICA, Campinas, São Paulo, 10.I.2006.
	Solanum gilo Raddi (scarlet eggplant), Pipiripau, Planaltina, Distrito Federal,
	22.XI.2005 and Caturra farm, Vale do Ribeira, Miracatu, São Paulo, 16.I.2006.
	Caturra farm, Miracatu, Vale do Ribeira, São Paulo, 16.I.2006.
	This is the first report of T. desertorum on eggplant (S. Melongena) in South
	America. It was registered previously in Japan by

[START_REF] Baker | Arañas rojas de América Central (Acarina:Tetranychidae)[END_REF] 

reported T. desertorum on M. pruriens in Central America. Therefore, M. aterrima is a new registered host in the Fabaceae family for the T. desertorum in South America.

Magnoliaceae

Michelia champaca L. (yellow magnolia), Faculdade da Terra de Brasília, Recanto das Emas, Distrito Federal, 28.XI.2005 and the Universidade Federal de Lavras, Lavras, Minas Gerais, 06.V.2006. Michelia champaca is a new host for T. desertorum. Solanaceae Solanum melongena L. (eggplant), Faculdade da Terra, Recanto das Emas,

Distrito Federal, 28.XI.2005. 

  described this species from samples collected from soursop (Annona muricata) in the state of São Paulo. Its presence on A. squamosa in the municipality of Mocambinho, in northern Minas Gerais, indicate a new locality for this specie in Brazil.

  emphasized the need to avoid introducing European mites into Brazil, and stated that the reason this specie was not present on grape vines in Brazil was unknown. They argued the possibility of biological differences between Brazilian and European mites.The spread of P. ulmi infestation on grape vines from Rio Grande do Sul(2005/2006) to Minas Gerais (2006) state could be result of the two states trading plant propagation material. The hypothesis that European mites were introduced into Brazil cannot be discarded considering that P. ulmi were discovered approximately 40 years ago and have only recently been causing damage to grape vines.[START_REF] Ferla | Ocorrência do ácaro vermelho europeu associado à cultura da videira no Rio Grande do Sul, Brasil[END_REF] state that the probable causes for P. ulmi dispersion in Brazil are: the proximity of apple tree plantations to vineyards in the south of Brazil; trading infested plant material within Brazil and abroad; and unbalance orchards due to excessive chemical products, especially non-selective fungicides.

Conclusions

Thirty-five new hosts were registered for 11 tetranychid species already known in Brazil: E. banksi (6 hosts), M. tanajoa (2), O. anonae (1), O. mangiferus (3), T. bastosi (1), T. desertorum (4), T. evansi (1), T. ludeni (4), T. mexicanus (2), T. neocaledonicus (3), and T. urticae (8), as well as one in Uruguay for T. urticae. New localities were registered in Brazil for E. tremae, O. anonae, T. gloveri and P. ulmi.

Table 1 .

 1 Collection records of Tetranychus urticae mites sequenced in this study

	Code	Country	Locality		Host Plant		Latitude	Longitude	GenBank	GenBank
				Scientific name	Family	Common name			accessions ITS	accessions COI
	* REF France	France	Elne	Nerium oleander	Apocynaceae Oleander	42º 35' 57.28" N 02º 57' 59.02" L	HM565873	HM565891
	* 6_1 France	France	Elne	Nerium oleander	Apocynaceae Oleander	42º 36' 33.02" N 02º 58' 05.68" L	HM565874	HM565892
	* 12_1 France	France	Perpignan	Datura stramonium	Solanaceae	Jimsonweed	42° 42' 05.66" N 02° 53' 38.9 4" L	HM565875	HM565893
	* 8_2_07 Spain	Spain	S.Susanna/Cami de la Riera	Solanum melongena	Solanaceae	Eggplant	41° 38' 26.50" N 02°43' 00.74 " L	HM565876	HM565894
	* 24_3 France	France	Saint Nazaire	Datura stramonium	Solanaceae	Jimsonweed	42° 40' 09.61" N 02° 59' 27.4 4" L	HM565877	HM565895
	* 116_2 France	France	La Gaude-Bord du Var	Urtica dioica	Urticaceae	Stinging nettle	43º 43' 17.95" N 07º09" 04.98" L	HM565878	HM565896
	* 120_1 France	France	Nice	Phaseolus vulgaris	Fabaceae	Bean	43º 42' 11.45" N 07º 15' 09,13" L	HM565879	HM565897
	* 41_2 Spain	Spain	Vilademuls/Galliners	Solanum lycopersicum Solanaceae	Tomato	42° 8' 57.084" N 02° 52' 26.26" L	HM565880	HM565898
	* 42_1 Spain	Spain	Bordils/Juià	Solanum lycopersicum Solanaceae	Tomato	42º 02' 12.70" N 02º 54' 54.18" L	HM565881	HM565899
	* Ej Spain	Spain	Bordils/Juià	Solanum lycopersicum Solanaceae	Tomato	42º 02' 12.70" N 02º 54' 54.18" L	HM565882	HM565900
	* 43_1 Spain	Spain	S. Andreu de Llavaneres	Solanum nigrum	Solanaceae	Black nightshade	41° 34' 20.52" N 02° 28 ' 56.52" L	HM565883	HM565901
	* 97_1 Canary	Canary Island	Santiago del Teide	Musa acuminata	Musaceae	Cavendish banana 28º 17' 46.04" N 16° 48' 52.26" O	HM565884	HM565902
	* 116_2 Canary	Canary Island	Granadilla de Abona	Solanum lycopersicum Solanaceae	Tomato	28° 04' 15.32" N 16° 33 '47.38" O	HM565885	HM565903
	8 119_2 Canary	Canary Island	Arico, Playa del Rio	Solanum lycopersicum Solanaceae	Tomato	28° 10' 39.92" N 16° 28' 45.96" O	HM565886	HM565904
	* 119_3 Canary	Canary Island	Arico, Playa del Rio	Solanum lycopersicum Solanaceae	Tomato	28º 10' 21.68" N 16º 28' 23.82" O	HM565887	HM565905
	* 128_2 Canary	Canary Island	Agüimes, Playa	Solanum lycopersicum Solanaceae	Tomato	27° 54' 14.80" N 15° 26' 44.32" O	HM565888	HM565906
	** 13_1 Greece	Greece	Ieraptera	Solanum melongena	Solanaceae	Eggplant	35º 01' 22.00" N 25º 44' 35.00" L	HM565889	HM565907
	** 175_1 Brazil	Brazil	Recife, Pernambuco	Carica papaya	Caricaceae	Papaye	07º 49' 25,32" S 34º 54' 59,79" O	HM565890	HM565908
	*	Red form							
	**	Green form							

Table 1 .

 1 Collection records of Tetranychus urticae mites sequenced in this study

	Code	Country	Locality		Host Plant		Latitude	Longitude	GenBank	GenBank
				Scientific name	Family	Common name			accessions ITS	accessions COI
	* REF France	France	Elne	Nerium oleander	Apocynaceae Oleander	42º 35' 57.28" N 02º 57' 59.02" L	HM565873	HM565891
	* 6_1 France	France	Elne	Nerium oleander	Apocynaceae Oleander	42º 36' 33.02" N 02º 58' 05.68" L	HM565874	HM565892
	* 12_1 France	France	Perpignan	Datura stramonium	Solanaceae	Jimsonweed	42° 42' 05.66" N 02° 53' 38.9 4" L	HM565875	HM565893
	* 8_2_07 Spain	Spain	S.Susanna/Cami de la Riera	Solanum melongena	Solanaceae	Eggplant	41° 38' 26.50" N 02°43' 00.74 " L	HM565876	HM565894
	* 24_3 France	France	Saint Nazaire	Datura stramonium	Solanaceae	Jimsonweed	42° 40' 09.61" N 02° 59' 27.4 4" L	HM565877	HM565895
	* 116_2 France	France	La Gaude-Bord du Var	Urtica dioica	Urticaceae	Stinging nettle	43º 43' 17.95" N 07º09" 04.98" L	HM565878	HM565896
	* 120_1 France	France	Nice	Phaseolus vulgaris	Fabaceae	Bean	43º 42' 11.45" N 07º 15' 09,13" L	HM565879	HM565897
	* 41_2 Spain	Spain	Vilademuls/Galliners	Solanum lycopersicum Solanaceae	Tomato	42° 8' 57.084" N 02° 52' 26.26" L	HM565880	HM565898
	* 42_1 Spain	Spain	Bordils/Juià	Solanum lycopersicum Solanaceae	Tomato	42º 02' 12.70" N 02º 54' 54.18" L	HM565881	HM565899
	* Ej Spain	Spain	Bordils/Juià	Solanum lycopersicum Solanaceae	Tomato	42º 02' 12.70" N 02º 54' 54.18" L	HM565882	HM565900
	* 43_1 Spain	Spain	S. Andreu de Llavaneres	Solanum nigrum	Solanaceae	Black nightshade	41° 34' 20.52" N 02° 28 ' 56.52" L	HM565883	HM565901
	* 97_1 Canary	Canary Island	Santiago del Teide	Musa acuminata	Musaceae	Cavendish banana 28º 17' 46.04" N 16° 48' 52.26" O	HM565884	HM565902
	* 116_2 Canary	Canary Island	Granadilla de Abona	Solanum lycopersicum Solanaceae	Tomato	28° 04' 15.32" N 16° 33 '47.38" O	HM565885	HM565903
	8 119_2 Canary	Canary Island	Arico, Playa del Rio	Solanum lycopersicum Solanaceae	Tomato	28° 10' 39.92" N 16° 28' 45.96" O	HM565886	HM565904
	* 119_3 Canary	Canary Island	Arico, Playa del Rio	Solanum lycopersicum Solanaceae	Tomato	28º 10' 21.68" N 16º 28' 23.82" O	HM565887	HM565905
	* 128_2 Canary	Canary Island	Agüimes, Playa	Solanum lycopersicum Solanaceae	Tomato	27° 54' 14.80" N 15° 26' 44.32" O	HM565888	HM565906
	** 13_1 Greece	Greece	Ieraptera	Solanum melongena	Solanaceae	Eggplant	35º 01' 22.00" N 25º 44' 35.00" L	HM565889	HM565907
	** 175_1 Brazil	Brazil	Recife, Pernambuco	Carica papaya	Caricaceae	Papaye	07º 49' 25,32" S 34º 54' 59,79" O	HM565890	HM565908
	*	Red form							
	**	Green form							

Table 2 .

 2 PCR and sequencing primers used to obtain nuclear ribosomal ITS and mitochondrial COI sequences of Tetranychus urticae.

	Fragment	Length	Position	Primer	Reference	Primers	Use
		(bp)					
	ITS	1200	3' of 18S	18S	Ben Ali et al. 2000	5'-AGAGGAAGTAAAAGTCGTAACAAG-3'	PCR and sequencing
			5' of 28S	28SC	Navajas et al. 1998	5'-ATATGCTTAAATTCAGCGGG-3'	PCR and sequencing
			5.8S	InternS	Navia et al. 2005	5'-GATCACTCGAATTACCAATCG-3'	Sequencing
			5.8S	InternSRev	Navia et al. 2005	5'-CGATTGGTAATTCGAGTGATC-3'	Sequencing
	COI	890	COI	C1J 1718	Simon et al. 1994	5'-GGAGGATTTGGAAATTGATTAGTTCC-3'	PCR and sequencing
			COI	773	Navajas et al. 1992	5'-TACAGCTCCTATAGATAAAAC-3'	PCR and sequencing
			COI	IntCOI	This study	5'-GGNGANCCTATTTTATATCAAC-3'	Sequencing
			COI	IntCOI Rev	This study	5'-GTTGATATAAAATAGGNTCNCC-3'	Sequencing

Table 5 .

 5 ITS2 (482 bp) and COI (390 bp fragment) sequences of Tetranychus retrieved from Genbank and identification of those deposited in the EMBL as members of T. urticae and T. cinnabarinus (Supplementary Material, Appendix 1 and 2).

		ITS2		COI		T. urticae	T. cinnabarinus
		Nr	(%)	Nr	(%)	ITS2	COI	ITS2	COI
	Number of sequences	105	100	137	100	32	50	29	30
	Misidentified	26	25	36	26	3	14	23	21
	Dubious	6	6	9	7	-	-	6	9
	Unpublish	42	40	32	23	19	3	10	11

  Table 2 and Figure2). A legal permit to collect zoological material for scientific purposes is required by the Brazilian Institute for the Environment and

	Renewable Natural Resources (IBAMA) and therefore, we requested and were
	issued permit number 231/2006 CGFAU/IBAMA.
	At least 50 individual mites were collected in each locality. Mites were
	preserved in 100% ethanol for molecular analysis and in 70% ethanol for
	morphological identification.

Table 2 .

 2 Collection records of Tetranychus urticae mites from Paleartic region and Canada sequenced and respective information on amplified region.

	Voucher	Country	Collection sites
	Nr		

Table 4 .

 4 Descriptive analyses and molecular diversity indices. Intraspecific information based on ITS1 and ITS2 regions of rDNA for Tetranychus urticae sequences.

	Region	sequences Nr of	(pb) sites Analysed	(bp) deletions Insertions	(bp) analysed Position	(bp) sites Invariable	(bp) sites Variable	site (bp) informative Parsimony	site (bp) Polymorphic	K	Pi
	ITS1	59	517	44	473	448	15	10	25	2,96 0,0063
	ITS2	82	488	34	454	443	9	2	11	0,32 0,0007

Table 5 .

 5 Descriptive analyses and molecular diversity indices. Intraspecific information based on sequences of the COI region of Tetranychus urticae specimens.

	Nr of sequences	Analysed sites (pb)	Insertions deletions (bp)	Invariable sites (bp)	Variable sites (bp)	Parsimony informative site (bp)	Polymorphic site (bp)	K	Pi	h	Hd
	20	955	196	742	42	15	57	9,9883 0,0125 8 0,7251
	57	390	80	310	10	30	40	11,401 0,0325 21 0,893

Chapter 4. Phylogeny, phylogeography and genetic structure in Tetranychus urticae Koch (Acari: Tetranychidae) populations inferred from ITS2 and COI DNA sequences. In progress.

CROFT, B.A.; HOYT, S. C. (1983) Integrated management of insect pests of pome and stone fruits. New York: Wiley Interscience. (454p.).

* * * * This paper corresponds to part of the thesis of the senior author, submitted to the Animal Biology graduate program of the Institute of Biological Sciences, Department of Zoology within the scope of a double doctoral degree program of University of Brasília and University of Montpellier II -program SIBAGHE, Specialty: Evolution, ecology, genetic resources and palaeontology.

Koch, C. L. (1836). Deutschlands Crustaceen Myriapoden und Arachniden, 1:8.

Acknowledgements

I must start by acknowledging my supervisors Maria Navajas and Ivone Rezende Diniz. With their enthusiasm and sound advice they helped to accomplish my PhD. Their comments and suggestions on this thesis were most valued.

Acknowledgements

The authors are grateful to José Francisco Montenegro Valls, Embrapa

Recursos Genéticos e Biotecnologia for the grass identification. To Paulo Roberto V. S. Pereira, Embrapa Trigo and Patrícia Drumond, Embrapa Acre for helping with the mite collections. To Francisco José Ferragut, Universidad Politécnica de Valencia,

Acknowledgments

The authors would like to thank Fábio Aquino Albuquerque, Embrapa -Centro Nacional de Pesquisa de Algodão, Campina Grande, PA; Adalton Raga and Mario E. Sato, Instituto Biológico de São Paulo, Campinas, SP; Marineide Rosa Vieira, Faculdade de Ciências Agronômicas, UNESP, Botucatu, SP; Lino Bittencourt Monteiro, Universidade Federal do Paraná, Paraná, RS; Manoel G. C. Gondin, Universidade Federal Rural de Pernambuco, Recife, PE; José Maria Milanez, Embrapa Trigo, Passo Fundo, RS; Empresa Caliman, Espírito Santo; Aloyséia Cristina Noronha, Embrapa Embrapa Amazônia Oriental, Belén, PA; Marcílio Thomazini, Embrapa Acre, Rio Branco, Acre; Marcone C. M. Chagas, Empresa de Pesquisa Agropecuária do Rio Grande do Norte, Natal, RN; Elton Lúcio de Araújo, Universidade Federal Rural do Semi-Árido, Mossoró, RN; Ricardo Adaime da Silva, Embrapa Agroflorestal, Macapá, AP; Paulo E. Degrande, Universidade Federal da Grande Dourados, Dourados, MS; Marcelo Poletti, PROMIP Ltda, Piracicaba, SP and the students Renato Tratch and and Samuel Rogger for help in the collection of mite samples. Research for this paper was conduct in partial fulfillment of the requirements for the PhD degree of the senior author in Animal Biology at the University of Brasília and in Ecology, Biodiversity and Evolution at the University of Montpellier II. This work was partially supported by CAPES, Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil. The authors are also greateful to CNPq, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil, for the fellowship. Acknowledgements The authors thank Marc Maraun for valuable comments on the manuscript, Alain Migeon, for help in mite collections and mite identification, Anastasia Tsagkarakou, for providing samples from Greece and Sandrine Cros-Arteil, for technical assistance in molecular techniques. Research for this paper was conduct in partial fulfillment of the requirements of the first author, for a PhD degree in Animal Biology at the University of Brasília and in Ecology, Biodiversity and Evolution at the University of Montpellier II. This author thanks the members of the PhD committee: Denis Bourguet, Elisabeth Fournier, George Roderick, Isabelle Olivieri and Benoît Facon for valuable suggestions on the manuscript. This work was partially supported by CAPES, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil. The authors are also greateful to CNPq, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil, for the fellowship. Data used in this work were (partly) produced through molecular genetic analysis technical facilities of the IFR119 « Montpellier Environnement Biodiversité ».

Tetranychinae Berlese, Eurytetranychini Reck

Eutetranychus banksi (McGregor, 1914) Tetranychus banksi, McGregor, 1914. Type-host: Ricinus communis L. Type-locality: Orlando, Florida, United States.

Fabaceae

Acacia sp. (acacia), Universidade Federal Rural de Pernambuco, UFRPE, Recife, Pernambuco, 11.VIII.2006. Mucuna aterrima Holland (black mucuna), Instituto Agronômico de Campinas, ICA, Campinas, São Paulo, 10.I.2006. Phaseolus lunatus L. (lima beans), Embrapa Hortaliças, Gama, Distrito Federal, 07.XII.2005.

The presence of E. banksi on Acacia sp. was previously reported in Colombia (Urueta, 1975). The occurrence on this host from Pernambuco is the first to Brazil. McGregor (1914) described E. banksi on M. pruriens in the United States and Livshits and Salinas-Croche (1968) located it on Mucuna sp. in Cuba. Garret et al. (1967) reported E. banksi infestations on P. lunatus in Hawaii. This is the first report of E. banksi on M. aterrima and P. lunatus in South America. Magnoliaceae Michelia champaca L. (yellow magnolia), Faculdade da Terra, Recanto das Emas, Federal District, 28.XI.2005. Passifloraceae Passiflora edulis Sims (sour passion fruit), Lagoa Grande, Minas Gerais, 07.IV.2006. The Michelia champaca and P. edulis are new hosts for E. banksi. Solanaceae Solanum gilo Raddi (scarlet eggplant), Pipiripau, Planaltina, Distrito Federal, 22.XI.2005 and Caturra farm, Vale do Ribeira, Miracatu, São Paulo, 16.I.2006. Caturra farm, Miracatu, Vale do Ribeira, São Paulo, 16.I.2006. Solanum melongena L (eggplant), Faculdade da Terra, Recanto das Emas, Federal District, 28.XI.2005.

EC Experts' Group, Parma, Italy, October, 26-28 (pp. 90-101). Published for the Commission of the European communities by A. A. Balkema, Rotterdan, Boston. Gupta SK, Gupta YN. 1994. A taxonomic review of Indian Tetranychidae (Acari: Prostgmata) with description of new species, redescriptions of known species and keys to genera and species. Memoirs of the Zoological Survey of India 18(1): 1-196. Gupta SK. 1976. Contribution to our knowledge of tetranychid mites (Acarina) with descriptions of three new species from India. Oriental Insects 10(3): 327-351. Gupta SK. 1992. Arachnida: plant mites (Acari). In: Ghosh AK, editor. Fauna of West Bengal, (Arachnida and Acari) Part 3: 61-211. Calcutta, India: Zoological Survey of India. Gutierrez J, Schicha E. 1983. The spider mite family Tetranychidae (Acari) in New South Wales. International Journal of Acarology 9(3): 99-116. Hatzinikolis EN. 1969. Preliminary notes on Tetranychoid and Eriophyid mites infesting cultivated plants in Greece. Proceedings of the 2nd International Congress of Acarology 161-167. Helle W, Pijnacker LP 1985. Spider mites: their biology, narural enemies and control. World Crop Pests. vol. 1A & 1B, Amsterdam, The Netherlands: Elsevier Science Publishers. Ho CC, Lo KC. Chen WH. 1997. Spider mite (Acari: Tetranychidae) on various crops in Taiwan. Journal of Agricultural Research of China 46 (4): 333-346. Jeppson LR, Keifer HH, Baker EW. 1975. Mites injurious to economic plants. Berkeley: University of California Press. Livshits IZ, Salinas-Croche A. 1968. Preliminares acerca de los acaros "Tetranicos" de Cuba. Cuba, La Habana: Centro National Fitosanid. McGregor EA. 1914. Four new tetranychids. Annals of the Entomological Society of America 7: 354-364. McGregor EA. 1950. Mites of the family Tetranychidae. American Midland Naturalist 44(2): 257-420. Meyer MKPS, Ryke PAJ. 1959. A revision of the spider mites (Acarina: Tetranychidae) of South Africa, with descriptions of a new genus and new species. Journal of the Entomological Society of Southern Africa 22(2): 330-366.

PART -II:

 Chapters 1, 2, 3 and4for valorous suggestions on the elaboration of micrographs. This work was partially supported by CAPES, Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil. The authors are grateful to CNPq, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil, for the authors' fellowships.

Supplementary Material, Appendix 1. Tetranychid mites species collected in Brazil between 10/2004 and 07/2008 with information on collection sites, host plants, collecting dates and collectors. F-34988 Montferrier-sur-Lez cedex, France. E-mail: auger@supagro.inra.fr 94ºC for 3 min and then PCR was carried out for 35 cycles of 1 min denaturation at 94 ºC, 1 min annealing at 51 ºC and 1.5 min extension at 72 ºC. PCR products (5 µl) were visualized on a 1% agarose gel saturated with ethidium bromide in 0.5x TBE buffer (45mM tris base, 45 mM boric acid and 1mM EDTA, ph 8.0). In addition to the forward and reverse primers 18S and 28SC used to amplify the ITS region as described in Navajas et al. (1998), two internal primers were used to sequence the complete ITS region (InternalS and InternalSRev). These primers were defined in highly conserved 5,8S and 28S flanking the ITS2 region as previously published in Navia et al. (2005). The primers used to amplify the COI fragment were C1J1718 and 773 (Navajas et al. 1992;[START_REF] Simon | Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers[END_REF].

Additional primers used for sequencing the COI fragment were IntCOI and IntCOIRev. All primers are given in Table 2. Amplified fragments (ITS and COI) were directly sequenced both strands using an ABI 3730 automated DNA sequencer (Applied Biosystems). One specimen from each of the localities sampled was analyzed.

Sequence retrieval and dataset

The studied dataset was constructed with the 18 sequences of the two forms of T. urticae generated in this study (accession numbers Table 1) plus all available ITS2 and COI sequences available in Genbank (on June, 2008) deposited as T. urticae and T. cinnabarinus. We also added into the dataset the available ITS2 and COI sequences of closely related species of Tetranychus s. str. of the group 9 (Flechtmann and Knihinicki 2002) whose identification requires the expertise of a specialized taxonomist. These are: T. ezoensis Ehara, 1962, T. kanzawai Kishida, 1927, T. neocaledonicus André, 1937, T. parakanzawai Ehara, 1999, T. phaselus Ehara, 1960, T. piercei McGregor, 1950, T. pueraricola Ehara and Gotoh, 1996, T. truncatus and T. turkestani Ugarov and Nikolski, 1937. Because of similar red body color of other Tetranychus s. str. that can be misidentified with some of the latter mentioned species, we also included to the dataset the sequences available in Genbank of the following Tetranychus s. str. species: T. evansi [START_REF] Baker | The tetranychoid mites of Africa[END_REF], T. gloveri Banks, 1900, T. ludeni Zacher, 1913and T. okinawanus Ehara, 1995.

Results and discussion

Mite species identification of field samples

Morphological species identification revealed samples consisted of T. urticae specimens only, except in Brazilian samples. Several samples of soybean from Brazil were infested by the following mites associations: specimens of T. urticae and Mononychellus planki McGregor, 1950;T. desertorum Banks, 1900 and T. gigas Pritchard and Baker, 1955; or T. desertorum and M. planki. Likewise, T. urticae and M. tanajoa [START_REF] Bondar | Notas entomológicas de Bahia III[END_REF] were found together on the same cassava plant and T. ludeni and T. urticae on a bean plant. Thus, to get reliable results, only samples consisting of exclusively T. urticae were further studied.

Retrieved sequences and dataset

Alignment and comparison of sequences clearly excluded any potential confusion between species of the Tetranychus s. str. of the ninth group and the other red colored Tetranychus considered in this study (listed in material and methods).

Thus the latter were excluded from further analyses. Phylogenetic analyses presented below, uncovered that some sequences having been attributed to T. urticae and T. cinnabarinus (red form of T. urticae) in Genbank, felt in clades consisting on either sequences of T. kanzawai, T. truncatus, or T. turkestani (COI sequences), or these species plus T. pueraricola (ITS2 sequences). As a consequence the results discussed below concern these species only.

From a total of 105 ITS2 sequences and 137 COI sequences retrieved from Genbank, respectively 32 and 50 were from T. urticae and 29 and 30 from T. cinnabarinus. In addition, available sequences of the closely related species of the ninth species-group of Tetranychus s. str. were retrieved: T. kanzawai, (4 ITS2, 42 COI), T. truncatus (4 ITS2, 4 COI), T. turkestani (7 ITS2, 7 COI) and T. pueraricola (2 ITS2). All these accessions together with the 18 sequences of T. urticae generated in this study served to create a dataset of a total of 123 ITS2 and 155 COI sequences. For retrieved Genbank sequences, accession numbers, assigned species names, information about host plants (common and family names), sample localities, the size of base pairs and original bibliographic references see Supplementary Material, Appendix 1 for ITS and Appendix 2 for COI.

Different primer combinations were used to generate Genbank sequences in the various studies producing dissimilarities in size and the gene portion of the sequenced fragment. Our study focused on the ITS2 region which was presented in all the accessions and allowed a final length for all the sequences in the dataset of about 482 base pairs (bp). In the case of the COI gene, a central fragment of 390 bp was present in the majority of accessions and was selected for subsequent analysis. Sequences containing several ambiguities in this COI region [AF131105 (T. urticae), AF131106 (T. cinnabarinus), AF131107 and AY750704 (T. kanzawai), AF131108 (T. truncatus)] were excluded from the analyses. After such adjustments, the alignments of sequences were straightforward and implied few insertions/deletions for ITS2 sequences alignment only. When identical sequences were found several times in either Genbank or in those obtained in this study, a single sequence was included in the analysis and the number of times it was found was indicated between parentheses in further results (Figs. 1, 2 and3). The assigned haplotype number, the number of times found in the dataset and the respective Genbank accessions are presented in Table 3 for ITS2 and Table 4 for COI.

Phylogenetic position of ITS2 sequences

The resulting 28 different ITS2 aligned sequences (482 bp) (Table 3) were used to produce a Maximum Likelihood tree which includes 5 distinct clades (Fig. 1). The apparent polyphyly of some species results from misidentification of some of the accessions as discussed below for COI results, and the reliable sequences they included served to name the clades as: T. urticae, T. turkestani, T. pueraricola, T. kanzawai and T. truncatus. The 18 T. urticae sequences produced in this study (including green and red forms of this mite) collapsed into 4 different haplotypes (HAP2, HAP9, HAP10 and HAP11) which were identical or very close to most of the sequences from Genbank gathered in the clade T. urticae. HAP 2 consisting on sequences from France, Spain and Brazil and haplotypes 9, 10 and 11 from Spain, Greece and France, respectively. Genetic distances between T. urticae haplotypes ranged from 0.002 to 0.004. A high homogeneity of ITS2 sequences of T. urticae had already been reported using a larger sample of worldwide geographical origin (Navajas et al. 1998;Navajas and Boursot 2003).

A total of 26 sequences (8 haplotypes) retrieved from Genbank did not cluster in the expected clade. Three haplotypes having been deposited as T. urticae felt in the clade T. kanzawai (HAP 3, HAP 4, HAP 5) while five deposited as T.

cinnabarinus gathered with species of other taxa. Phylogenetic close but apart from T. urticae, appear the T. turkestani sequences. Sequences retrieved from Genbank as being T. pueraricola, T. kanzawai and T. truncatus are distributed in distinct clades in the phylogenetic tree. Noticeable, 23 of a total of 29 sequences named T. cinnabarinus on Genbank appear scattered in these 3 clades, all well supported in the tree with respectively 82%, 82% and 100% of bootstrap scores.

Six more identical sequences (HAP 15), having been deposited as T. cinnabarinus, were apart while close to the T. pueraricola sequences. It needs to be stressed that a single haplotype (HAP 12) is composed by sequences of specimens having been deposited in Genbank as T. truncatus and T. cinnabarinus. This group was fully supported by bootstrap score (100%). Likewise, one haplotype (HAP 5) gathers sequences having been deposited as belonging to three different species (T. urticae, T. cinnabarinus and T. kanzawai). All together these data strongly suggests that several ITS2 sequences found in Genbank and having deposited as T. urticae and T. cinnabarinus were erroneously named because of mite misidentification (Table 5). Considering these concerns it is not possible to make a definitive statement regarding the identity of these sequences.

Figure 2 summarizes sequences comparisons and stress inconsistency in species identification of nucleotide sequences in the litigious accessions (3 T. urticae and 23 T. cinnabarinus). When comparing T. urticae and T. kanzawai sequences (Fig. 2A), the same six diagnostic positions (79, 309, 339, 406, 411 and 454) between these two species are also present in some of the sequences deposited as T. cinnabarinus [START_REF]Among the pairs sampled, approximately 20% indicated a high level of distance (≥ 6%)[END_REF] and T. urticae (3). Similarly, nine diagnostic sites appear among T. urticae and T. truncatus sequences (68, 305, 309, 311, 321, 337, 339, 411 and 455; Fig. 2B) and seven among T. urticae and T. pueraricola (337 338,311,336,343,415 and 458;Fig. 2C). However, some T. cinnabarinus or T. urticae sequences from Genbank inconsistently present the same nucleotide at these diagnostic positions. When comparing T. urticae and T. turkestani sequences retrieved from Genbank plus one new produced in this work, it appears the same three diagnostic sites (339, 411 and 455) (Fig. 2D) reported by Navajas and Boursot (2003).

Phylogenetic information content of COI sequences

Consistently with the ITS2 results, the Maximum Likelihood phylogeny based on multiple alignments of the 58 mtCOI haplotypes (Table 4) obtained from Tetranychus species, revealed that most of the COI sequences having been deposited as T. cinnabarinus in Genbank (21 out of 30) branched together with sequences either one of the four well supported clades, named T. kanzawai, T. truncatus, T. turkestani and T. urticae in the tree (Fig. 3). Two haplotypes (HAP 33 and HAP 34) are apart (90 % of bootstrap support) and highly divergent to all the other analyzed sequences (8.7% to 12.6% of genetic distance: Kimura's twoparameter correction). These haplotypes probably belong to a different taxon.

Interestingly, the same mite samples having been used to produce HAP 33 and HAP 34 sequences of the COI, were used to generate HAP 16 of the ITS2 (Supplementary Material, Appendix 3) which grouped together with T. pueraricola sequences (Fig. 1). While the related publication [START_REF] Xie | Genetic relationship between the carmine spider mite Tetranychus cinnabarinus (Boisduval) and the two-spotted mite T. urticae Koch in China based on the mtDNA and rDNA ITS2 sequences[END_REF]) doesn't allow to verify if the same mite individual were used to produce both COI and ITS2 sequences, it seems plausible that COI haplotypes HAP33 and HAP 34 correspond to T. pueraricola. Five other T. cinnabarinus haplotypes (HAP 38,39,40,41,56) are clearly apart (100%) and far from the rest of the sequences here examined [10 to 11.9 of genetic divergence to the sequences in other clades]. The identity of these accessions remains unsolved.

Table 3. Accession numbers of ITS2 sequences retrieved from Genbank for a set of Tetranychus species. The haplotype number attributed in this study (see texte) and the respective species identification in Genbank are indicated. The sequences obtained in this study were included. * The number in parentheses indicates the number of times a haplotype was found in the dataset. For example: 1 (x2) means that Haplotype 1 was found two times. The complete list of the Tetranychus COI sequences from Genbank with information on host plant species, geographic origin and references are in Supplementary Material, Appendix 1. ** Sequences obtained in this study are in bold.

Haplotype number

Accessions number in GenBank and the sequences obtained in this study Species name in GenBank (x 2)* X99881, AY750700 T. urticae

AB076369, AM408030, AM408045, AM408031, AM408043, AM408035, AM408044, AM408046, DQ656440, DQ680165, DQ680166, DQ680167, DQ680168, DQ680169, DQ680170, DQ680171, DQ778064, DQ778065, DQ778066, DQ778067, DQ778068, DQ778069, GQ141923, GQ141924, REF_FR, 6.1_FR,8.2_07_ES,12.1_FR,24.3_FR,41.2_ES,42.1_ES,43.1_ES,116.2_CANARY,119.2_CANARY,119.3_CANARY,120.1_FR,128.2_CANARY,175 

T. urticae T. urticae T. urticae T. pueraricola

A.

4 5 7 2 3 3 3 4 4 4 4 4 6 8 9 3 0 3 8 0 1 2 3 5 1 9 9 8 6 1 0 4 

HAP T. urticae (39 x) A G G A C G G C A G -A HAP T. kanzawai . T A

B. Haplotype number and species name in Genbank

Variables sites Species 6 9 1 2 3 3 3 3 3 3 3 4 4 8 4 2 1 0 0 1 2 3 3 9 1 5 0 0 5 9 1 1 7 9 5 1 0 1 1 1 2 3 3 3 3 3 4 4 4 0 5 6 2 1 3 3 3 4 1 5 9 9 3 3 5 1 6 7 8 3 5 

HAP T. urticae (39 x) T T C A T C G C T T C A A HAP T. truncatus C . . G C A A A G A T G T HAP T. Truncatus

C. Haplotype number and species name in Genbank Variable sites Species

HAP T. urticae (39 x) C T --C C T --G A A HAP T. pueraricola T . --G A C A C A T T HAP T. pueraricola T Y A A . A C A C A T T HAP T. cinnabarinus(3 x)

. .

--. A C A C A T T

D. Haplotype number and species name in GenBank

Variable sites Species 1 3 3 3 3 4 4 4 4 5 1 0 3 5 6 1 3 4 5 8 6 9 9 8 7 1 4 6 T. truncatus

T. urticae

One cluster (named T. truncatus) groups all the sequences retrieved from Genbank having been identified as T. truncatus (99% of bootstrap support) but includes also 14 sequences having been deposited as T. urticae (and 13 deposited as T. cinnabarinus). Most of these sequences correspond to specimens collected from China [START_REF] Xie | Population structure of the two-spotted spider mite (Acari:Tetranychidae) from China[END_REF][START_REF] Xie | Genetic relationship between the carmine spider mite Tetranychus cinnabarinus (Boisduval) and the two-spotted mite T. urticae Koch in China based on the mtDNA and rDNA ITS2 sequences[END_REF]. The identification of 12 sequences as T. urticae had already been questioned by Hinomoto et al. (2007). The authors suggested that these sequences likely were T. truncatus because they were quite different from T. urticae sequences but highly similar to COI sequences they produced from Japanese T. truncatus specimens. Likewise, other two sequences deposited as T. urticae (unpublished data) and the 13 sequences deposited as T.

cinnabarinus [START_REF] Xie | Genetic relationship between the carmine spider mite Tetranychus cinnabarinus (Boisduval) and the two-spotted mite T. urticae Koch in China based on the mtDNA and rDNA ITS2 sequences[END_REF]) and included in the same cluster, could also have The presence of T. turkestani sequences also included in this cluster is not surprising. According to Navajas and Boursot (2003), while ITS sequences clearly separated T. urticae and T. turkestani in two distinct taxa, the speciation of the two mite species is recent enough that the segregation of mtCOI lineages has not occurred, and this sister species has not yet reached monophyly for mtDNA.

* The number in parentheses indicates the number of times a haplotype was found in the dataset. For example: 1 (x13) means that the Haplotype 1 was found thirteen times. 

T. kanzawai T. truncatus T. urticae & T. turkestani

DNA sequences a valuable source of information needing cautiously taxonomic analysis

The misleading species assignment of sequences in a group of closely related mite species, as uncovered by this study, highlights the strong need for an accurate determination of the species identity which should be supervised by specialists of the studied group. As for other minute arthropods, many mite species have a reduced number of morphological characters to be used in taxonomy, and in the case of plant mites, as the Tetranychidae, two main additional reasons may cause wrong taxa identification. First, in the field, plants can be plagued by several mite species at the same time as was observed during the identification of a few mites samples collected for this study and reported by Foott (1962Foott ( , 1963)). A second cause of equivocal identification of spider mites is the difficulty to find adult males in field samples, because the female-biased sexratio which often characterizes these mites. However, species discrimination of most spider mites and in particular those of the group 9 considered in this study can only be achieved by the observation of the shape of the aedeagus, which is the external part of the male genitalia. As this character is essential for species discrimination, the absence of male specimens from a sample makes specific identification of mites belonging to the species-group 9 impossible.

The controversial validity of the taxon T. cinnabarinus (considered by many authors as the red form of T. urticae), which has been questioned for longtime in the specialized literature, is an example of the difficulty to identify spider mites species and its consequence in database solidity. A great confusion still continues on the validity of T. cinnabarinus. Despite the work by Dupont (1979), who showed that the green and the red forms can interbreed, this taxon still remains considered as valid by a few authors (e. g. cinnabarinus. In addition, the field samples of T. urticae collected in this study, support the results obtained by Hinomoto et al. (2001) coming to the synonymy between T. urticae and T. cinnabarinus (Gotoh and Takioka 1996;Bolland et al. 1998;Ehara 1999). Likewise, in this study sequences of T. urticae group together in a same clade in both the ITS2 and the COI phylogenetic trees, providing additional support to the invalidity of the T. cinnabarinus taxon.

There is increasing evidence from other taxa that COI is not an appropriate gene for barcoding in many invertebrates. Remarkable degree of COI divergence was observed, for instance, in the Opiliones, Aoraki denticulata (Foster, 1948), commonly known as daddy long-legs or harvestmen [START_REF] Boyer | Deep Genetic Divergences in Aoraki Denticulata (Arachnida, Opiliones, Cyphophthalmi): a widespread "mite harvestmen" defies DNA taxonomy[END_REF]. Therefore, some of the COI sequences in the databank may be wrong but some may also reflect the large variation of this gene within species. In our study we found evidences of misidentification among databank sequences of ninth species-group of Tetranychus s. str. showed in phylogenies based on two different markers (COI and ITS).

DNA sequences already have an important role in systematic and routine identifications in spider mites (e.g. as for the barcode initiative), and no doubt that this will play and increasing role in the future to address a large palette of questions. For that reason, it is crucial that wrong species identification of the associated sequences does not contaminate the valuable resource that public databases represent. This raises the question as to what extend these sequences can be used as reliable but also highlights the importance of careful sampling and detailed morphological identification of field collected specimens as a basic process to subsidize molecular biology studies. The preservation of voucher specimens encompassing public DNA sequences appears as a necessary requirement to allow solid comparative molecular studies. 

Scientific article in progress

ABSTRACT

The two-spotted spider mite Tetranychus urticae Koch is a highly polyphagous species which damages many crops worldwide. Studies on the genetic variability of T. urticae populations were conducted in France, Spain, Israel and Japan, although this spider mite produces outbreaks in many other countries, including Brazil, where it is considered one of the most important pest among the phytophagous mites. This study investigates genetic variability, phylogenetic relationships and and eventual host plant associations of T. urticae populations collected around the world. Two molecular markers were used: ITS (ribosomal DNA) and COI (mitochondrial DNA). New sequences of these both regions were obtained from individuals collected in Brazil and Palearctic Region (France, continental Spain, Canary Islands, Greece, Syria, Tunisia, Poland and Norway and Canada) and were jointly analyzed with the T. urticae sequences deposited in GenBank. Specimens were directly mounted on microscopic slide preparations to confirm the species identity by morphological analysis before molecular analyses.

Results indicated the occurrence of genetic structure of the analyzed T. urticae populations according to its geographic collection locality. However, any effect of the host plant was observed. Sequence diversity inferred from both studied genomic regions (ITS and COI) was higher among the Mediterranean countries. Genetic variability accessed from ITS region was not observed among populations from different regions in Brazil. Two COI haplotypes were found in Brazilian populations, one of them also present in France, Spain and the Canary Islands and the other in Japan.

Key words: DNA sequences, genetic variability, mitochondrial DNA, ribosomal DNA, molecular markers, two-spotted spider mite, Brazil 

A.

microcentrifuge tube adding 50 µl of ultrapure water on the membrane of the mini spin column.

Two target DNA fragments were PCR-amplified and sequenced per single mite: the nuclear ribosomal region spanning the ITS1, 5.8S and ITS2 region (about 1200 bp) and a fragment of the mitochondrial COI gene (about 950 bp). The amplification reactions were carried out in 25 µL volumes containing 2.5 µl of 10x buffer supplied by the manufacturer, 0.13 un/µL (5 units) of Taq polymerase (Qiagen), 2 µL DNTP (0.25 mM of each base), 0.5 µM of each oligonucleotide primer (10 mM), 2 µl of MgCl 2 (25 mM), 15.37 µL or 13.37 µL water and 2 or 4 µL of the template DNA for ITS and COI, respectively. Samples were denatured at 94ºC for 3 min and then PCR was carried out for 35 cycles of 1 min denaturation at 94 ºC, 1 min annealing at 51 ºC and 1.5 min extension at 72 ºC. PCR products (5 µl) were visualized on a 1% agarose gel saturated with ethidium bromide in 0.5x TBE buffer (45mM tris base, 45 mM boric acid and 1mM EDTA, ph 8.0).

In addition to the forward and reverse primers 18S and 28SC used to amplify the ITS region as described in Navajas et al. (1998), two internal primers were used to sequence the complete ITS region (InternalS and InternalSRev).

These primers were defined in highly conserved 5,8S and 28S flanking the ITS2 region as previously published in Navia et al. (2005). The primers used to amplify the COI fragment were C1J1718 and 773 (Navajas et al. 1992;[START_REF] Simon | Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers[END_REF].

Additional primers used for sequencing the COI fragment were IntCOI and IntCOIRev. All primers are given in Table 3. Amplified fragments (ITS and COI) were directly sequenced both strands using an ABI 3730 automated DNA sequencer (Applied Biosystems). One specimen from each of the localities sampled was analyzed.

Sequence retrieval and dataset

The studied dataset was constructed with the 56 ITS and 20 COI sequences of T. urticae generated in this study according the collection data (Table 1 and2) plus all available ITS (27) and COI (37) sequences in Genbank (on June, 2008) deposited as T. urticae (Supplementary Material, Appendix 1 for ITS and 2 for COI sequences).

Sequence and phylogenetic analyses

Sequences were aligned using the CLUSTAL W Multiple alignment procedure (Thompson et al. 1994) implemented in the BIOEDIT software, version 7.0.4 (Hall 1999). No manual adjustments to the CLUSTAL alignment were performed.

Intraspecific measures of DNA sequences variation were assessed using DnaSP software version 4.50 [START_REF] Rozas | DnaSP, DNA polymorphism analyses by the coalescent and other methods[END_REF] and were estimated for both ITS and COI sequences alignments: nucleotide polymorphism; number of variable sites; polymorphic sites; parsimony informative sites; nucleotide diversity, Pi (π)the average number of nucleotide differences per site between two sequences; the average number of nucleotide differences (k) [START_REF] Tajima | Evolutionary relationship of DNA sequences in finite populations[END_REF]; haplotype numbers and distribution and haplotype diversity (h).

Pairwise nucleotide distances were calculated using Kimura's twoparameter correction model and the software MEGA 4 (Kimura 1980;Tamura et al. 2007) for ITS2 and COI sequences of T. urticae considered in this work.

Phylogenetic relationships were estimated based on both ITS2 and COI sequence alignments using the Neighbour Joining (NJ) method based on sequence distances calculated using the Kimura's two-parameter correction method (K2P). For the NJ analyses robustness of nodes was assessed with 1000 bootstrap replication.

Analysis of Molecular Variance (AMOVA) and genetic structure accessed by COI sequences of Tetranychus urticae

The genetic differentiation on T. urticae populations was examined using COI sequences variance. The dataset was constructed with 20 new sequences obtained in this study together with 37 T. urticae sequences available in GenBank, totalizing 57 sequences (Table 1 and2, Supplementary Material, appendix 2).

The partition of the genetic variation among and between T. urticae populations from different regions as well as by different host plants was explored by hierarchical Analysis of Molecular Variance (AMOVA), as implemented in Arlequin software version 3.1 [START_REF] Excoffier | Analysis of molecular variance inferred from metric distances among DNA haplotypes: Application to human mitochondrial DNA restriction data[END_REF][START_REF] Excoffier | Arlequin ver. 3.0: An integrated software package for population genetics data analysis[END_REF] ) (see the 6). The Neighbor-Joining (NJ) method was used based on distances calculated using Kimura's two-parameter (K2P) correction. Bootstrap values (higher than 50%) are near to the branches. The scale bar indicates the number of changes per site in NJ(K2P) inference. Differentiation for all pairs of samples were not significant. Even considering geographically close population groups, the effect of the host plant on the genetic differentiation was not confirmed. Tetranychus urticae collections on Rosaceae and Caryophyllaceae families from Japan were held in close cities as Tsukuba, Tomobe (Honshu, Ibaraki) and Inzai (Honshu, Chiba) so the geographic effect was excluded supporting the results of genetic differentiation related to host plant family in Japan. [START_REF] Nishimura | The genetic variation among populations of Tetranychus kanzaway complex (Acari: Tetranychidade) indicating by using mitochondrial, ribosomal, and microsatellite DNA markers[END_REF] studied the genetic structure of seven Japanese populations of T.

kanzawai, a closely related species of T. urticae, reported genetic structure within population related to the host plant effect. The authors found no positive correlation between geographic distance and differentiation between T. kanzawai populations. Consider the geographical size under study when assessing the geographic factor on structuring of T. urticae population is important because the distances may affect the genetic differentiation between populations.

The AMOVA results when adding T. urticae populations from Brazil and the Netherlands to those from Mediterranean and Asian regions were significant, with 53.57% of genetic variation between populations (Fst = 0.5357, p = 0). The Exact Test of Sample Differentiation (Table 9) showed that populations from Asian region are significantly different (≤ 0.05) from those of the other three regions (Mediterranean, Brazil and Netherlands). These results are exploratory for Brazil and the Netherlands, since only two populations were collected for each of these countries. Even though, the influence of the geographical effect on genetic structure of T. urticae populations was verified. The effect of the host plant, although not frequent, was detected in Japan 

Distribution and frequency of ITS2 sequences

Ten ITS2 different sequences were found among the 82 populations analyzed (Figure 4). Sequence type 1 presented a greater extent of geographic distribution and was the most frequent, representing 86.58% (71 sequences) of all sequences found in South America, Europe, the Macaronesian region, Asia, and Africa. It was the only sequence found in Brazil among the 22 populations sampled in the five regions of the country. In China, Japan, and Syria where 12, 20 and two sequences were respectively analyzed, and in Tunisia, Norway and Poland where only one sequence was analyzed, sequence type 1 was the only one recorded. In countries of the Mediterranean region -France and Spain -this sequence was the most frequent, representing around 85% of the sequences obtained. In the Canary Islands, sequence types 1 and 3 were found, and they were also present in peninsular Spain. Sequence type 3 was found only in these two places, while sequence type 7 was found in Holland and Taiwan.

The other sequence types were specific to particular countries: sequences 6 and 8 in Spain, sequences 4 and 5 in France, sequence 2 in Greece and sequences 9 and 10 in Israel. The greatest diversity of sequences was found among populations gathered in countries which border the Mediterranean Sea:

France (3 sequences types), Spain (4 sequence types), Greece (1) and Israel (2).

Distribution and frequency of COI haplotypes

Twenty-one COI haplotypes were found (Figure 5). Haplotype 1 was the most frequent, representing 22.81% of all haplotypes recorded, followed by 10 (19.29%), 8 (14.04%), 3 (5.26%) and 17 (5.26%). Haplotypes 1 and 10 were confirmed in Brazil and were also found in Asia (Japan, Taiwan, Thailand), in the Mediterranean region (Spain, France), and in the Canary Islands, respectively. It is important to note that only two T. urticae sequences were analyzed in Brazil.

Although small in sample size, the two most common haplotypes in the world (precisely haplotypes 1 and 10) were found in Brazil.

The presence of haplotypes 1 and 10 in the country is not surprising if viewed from a historical perspective. The age of exploration, the discovery of the Americas and the arrival of the Portuguese and Spanish in the Indies extended commerce to regions that were then regarded as remote. The Mediterranean Sea played a vital role in supporting navigation in the Atlantic Ocean. The Canary Islands served as a warehouse for commerce with Africa, America, Asia, and Europe to facilitate navigation and commerce along the routes at that time. East Asia was an important region for European expeditions searching for spices. This commerce stimulated a global redistribution of resources, including the diffusion of plants cultivated by mankind in various parts of the world. Wheat from the East Suppementary Material. Appendix 1. List of Tetranchus urticae ITS sequences retrieved from Genbank to construct the dataset. Accession numbers were copied from Genbank. Information about host plants (common and family names), sample localities, the size of base pairs and original bibliographic references are given. [START_REF] Ben-David | ITS2 sequences as barcodes for identifying and analyzing spider mites (Acari: Tetranychidae)[END_REF].

MAIN RESULTS

Chapter 1

Four tetranychid species collected during this research are new to science.

Two belong to the genus Oligonychus Berlese and were collected from grapes (Vitis (Navajas et al. 1998;Hinomoto et al. 2001). Likewise, ITS2 data from this study revealed the greatest diversity among populations gathered in countries on the Mediterranean basin (note that around 70% of new ITS2 sequences were added to the analyzed dadaset). ITS sequences produced from T. urticae populations from different regions in Brazil was completely homogenous. By contrast, two COI haplotypes were detected in Brazil. One of them was also present in France, Spain and the Canary Islands; and the other, in Japan.

An additional sampling effort, including samples from unexplored regions no doubt will strength our dataset. This is a long term project, but from now we are reviewing our publication strategy. While a paper draft has been already produced, we are considering to include additional microsatellite data (already produced but not yet analyzed), in order to increase the datset and to better support the findings on T.

urticae from Brazil.

CONCLUSION

Undoubtedly important studies on tetranychid mites have been conducted in several fields such as taxonomy and it has brought irrefutable advances to scientific knowledge available on this group of mites in Brazil. The taxonomic approach used in