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    Bayesian inference consensus of the combined analysis of sequences of Triatoma species focused on Triatoma jatai based on 16S and COI genes. Molecular evolution models for each partition were HKY + I + G for 16S (444 bp) and GTR + I + G for COI (492 bp). Numbers above the nodes indicate Bayesian posterior probabilities. Panstrongylus megistus was selected as out-group.

  • 1.

    Gonçalves TC, Teves-Neves SC, Santos-Mallet JR, Carbajal-de-la-Fuente AL, Lopes CM, 2013. Triatoma jatai sp. nov. in the state of Tocantins, Brazil (Hemiptera: Reduviidae: Triatominae). Mem Inst Oswaldo Cruz 108: 429437.

    • Search Google Scholar
    • Export Citation
  • 2.

    Obara MT, Barata JMS, da Rosa JA, Almeida PS, Gonçalves GA, Dale C, Gurgel-Gonçalves R, 2012. Description of the female and new records of Triatoma baratai Carcavallo & Jurberg, 2000 (Hemiptera: Heteroptera: Reduviidae: Triatominae) from Mato Grosso do Sul, Brazil, with a key to the species of the Triatoma matogrossensis subcomplex. Zootaxa 3151: 6368.

    • Search Google Scholar
    • Export Citation
  • 3.

    Schofield CJ, Galvão C, 2009. Classification, evolution, and species groups within the Triatominae. Acta Trop 110: 88100.

  • 4.

    Gardim S, Almeida CE, Takiya DM, Oliveira J, Araújo RF, Cicarelli RMB, da Rosa JA, 2014. Multiple mitochondrial genes of some sylvatic Brazilian Triatoma: non-monophyly of the T. brasiliensis subcomplex and the need for a generic revision in the Triatomini. Infect Genet Evol 23: 7479.

    • Search Google Scholar
    • Export Citation
  • 5.

    Justi SA, Russo CA, Mallet JR, Obara MT, Galvão C, 2014. Molecular phylogeny of Triatomini (Hemiptera: Reduviidae: Triatominae). Parasit Vectors 7: 149.

    • Search Google Scholar
    • Export Citation
  • 6.

    Sambrook J, Russell DW, 2001. Molecular Cloning: A Laboratory Manual, 3rd edition. New York, NY: Cold Spring Harbor Laboratory Press.

  • 7.

    Sainz AC, Mauro LV, Moriyama EN, García BA, 2004. Phylogeny of triatomine vectors of Trypanosoma cruzi suggested by mitochondrial DNA sequences. Genetica 121: 229240.

    • Search Google Scholar
    • Export Citation
  • 8.

    Hall TA, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41: 9598.

    • Search Google Scholar
    • Export Citation
  • 9.

    Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG, 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 29472948.

    • Search Google Scholar
    • Export Citation
  • 10.

    Gardim S, Rocha CS, Almeida CE, Takiya DM, da Silva MT, Ambrósio DL, Cicarelli RMB, da Rosa JA, 2013. Evolutionary relationships of the Triatoma matogrossensis subcomplex, the endemic Triatoma in central-western Brazil, based on mitochondrial DNA sequences. Am J Trop Med Hyg 89: 766774.

    • Search Google Scholar
    • Export Citation
  • 11.

    Huelsenbeck JP, Ronquist F, 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754755.

  • 12.

    Posada D, 2008. jModelTest: phylogenetic model averaging. Mol Biol Evol 25: 12531256.

  • 13.

    Mas-Coma S, Bargues MD, 2009. Populations, hybrids and the systematic concepts of species and subspecies in Chagas disease triatomine vectors inferred from nuclear ribosomal and mitochondrial DNA. Acta Trop 110: 112136.

    • Search Google Scholar
    • Export Citation
  • 14.

    Hypsa V, Tietz DF, Zrzavý J, Rego RO, Galvao C, Jurberg J, 2002. Phylogeny and biogeography of Triatominae (Hemiptera: Reduviidae): molecular evidence of a New World origin of the Asiatic clade. Mol Phylogenet Evol 23: 447457.

    • Search Google Scholar
    • Export Citation
  • 15.

    Monteiro FA, Costa J, Solé-Cava AM, 1998. Genetic confirmation of the specific status of Triatoma petrochii (Hemiptera: Reduviidae: Triatominae). Ann Trop Med Parasitol 92: 897900.

    • Search Google Scholar
    • Export Citation
  • 16.

    Abad-Franch F, Pavan MG, Jaramillo ON, Palomeque FS, Dale C, Chaverra D, Monteiro FA, 2013. Rhodnius barretti, a new species of Triatominae (Hemiptera: Reduviidae) from western Amazonia. Mem Inst Oswaldo Cruz 108 (Supp 1): 9299.

    • Search Google Scholar
    • Export Citation
  • 17.

    Almeida CE, Oliveira HL, Correia N, Dornak LL, Gumiel M, Neiva VL, Harry M, Mendonça VJ, Costa J, Galvão C, 2012. Dispersion capacity of Triatoma sherlocki, Triatoma juazeirensis and laboratory-bred hybrids. Acta Trop 122: 7179.

    • Search Google Scholar
    • Export Citation
  • 18.

    Alevi KCC, de Oliveira J, Moreira FF, Jurberg J, da Rosa JA, de Azeredo-Oliveira MTV, 2015. Chromosomal characteristics and distribution of constitutive heterochromatin in the Matogrossensis and Rubrovaria subcomplexes. Infect Genet Evol 33: 158162.

    • Search Google Scholar
    • Export Citation
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Mitochondrial Genes Reveal Triatoma jatai as a Sister Species to Triatoma costalimai (Reduviidae: Triatominae)

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  • Laboratório Interdisciplinar de Vigilância Entomológica em Diptera e Hemiptera, Instituto Oswaldo Cruz/Fundação Oswaldo Cruz, Rio de Janeiro, Brazil; Programa de Pós-Graduação em Biologia Animal, Universidade Federal Rural do Rio de Janeiro, Rio de Janeiro, Brazil; Departamento de Ciências Biológicas, FCF/UNESP, São Paulo, Brazil; Universidade Estadual Paulista “Júlio de Mesquita Filho”, São Paulo, Brazil; Laboratorio Ecoepidemiología, Instituto de Ecología, Genética y Evolución (IEGEBA–CONICET), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina; Programa de Pós-Graduação em Ecologia e Monitoramento Ambiental, Universidade Federal da Paraíba, Paraíba, Brazil

Triatoma jatai was described using a set of morphological structures from specimens collected in Paranã municipality of Tocantins State, Brazil. Under a Bayesian framework and using two mitochondrial genes (16S and COI), phylogenetic analysis recovered T. jatai as a sister species to Triatoma costalimai with higher genetic distances than between other well-recognized species. Our results agree with previous suggestions based on morphometric analysis. In the light of the non-monophyly of Matogrossensis subcomplex, the inclusion of T. jatai shall be considered for reevaluating this group.

The recently described Chagas disease vector, Triatoma jatai, occurs in rocky outcrops and currently presents its distribution restricted to the municipality of Paranã, Tocantins State, north of Brazil.1 The natural environment where T. jatai occurs is a target of continuous environmental degradation, such as deforestation and burning activities.

In T. jatai description, the authors observed a close relationship with Triatoma costalimai. These two species can be differentiated by the size, general color and shape of wings, connexivum, intersegmental sutures, and genital structures, which made it possible to refine the taxonomic key built in 20122 for the inclusion of T. jatai. In addition to these morphological similarities, they observed morphometric closeness, suggesting that it should be included in the Matogrossensis subcomplex of the Infestans complex, which previously included Triatoma baratai, T. costalimai, Triatoma deaneorum, Triatoma guazu, Triatoma jurbergi, T. matogrossensis, Triatoma vandae, and Triatoma williami.3

In the article that brought up T. jatai to science, Gonçalves and others1 evidenced its occurrence in sympatry with T. costalimai in limestone outcrops. A close relationship between these two species was also suggested by wing morphometrics.1 Thus, in this study, two mitochondrial genes (16S and COI) were sequenced and analyses were run under a Bayesian framework to evaluate the phylogenetic relationships between T. jatai and other South American triatomines.

The wild samples of T. jatai, T. costalimai, and Triatoma sordida used in this study are shown in Table 1 and their GenBank accession numbers in Table 2. Evaluate the phylogenetic position of T. jatai, we included species representing the six subcomplexes defined in 2009,3 whenever available in GenBank, we added samples from the same isolate. In addition, the choice of species downloaded from GenBank for running the analysis (Table 2) was based on the monophyletic clade of South American Triatoma.4,5 Panstrongylus megistus was set as the outgroup.

Table 1

Samples of triatomines used in this work

Subcomplex3SpeciesOrigin
MatogrossensisTriatoma jatai_03Paranã, TO
T. jatai_05Paranã, TO
T. jatai_16Paranã, TO
Triatoma costalimai_06Aurora do Tocantins, TO
T. costalimai_09Aurora do Tocantins, TO
Triatoma williami_04Barra do Garças, MT
T. williami_05Barra do Garças, MT
SordidaTriatoma sordida_PAR03Paranaíba, MS
T. sordida_ITA24Itaobim, MG

TO, MT, MS, and MG are Tocantins, Mato Grosso, Mato Grosso do Sul, and Minas Gerais states, respectively.

Table 2

Accession codes from GenBank sequences of Triatoma and out-group species used in the phylogenetic analysis

Subcomplex3Species16SCOI
BrasiliensisTriatoma brasiliensisKC248985KC249318
Triatoma sherlockiKC249068KC249377
InfestansTriatoma infestansKC249014KC249348
Triatoma platensisKC249363KC249047
Triatoma delponteiKC249332KC249001
MaculataTriatoma maculataAF324524AF449139
MatogrossensisTriatoma jatai_03KT601153KT601162
T. jatai_05KT601154KT601163
T. jatai_16KT601155KT601164
Triatoma costalimai_06KT601151KT601160
T. costalimai_09KT601152KT601161
Triatoma williami_04KT601156KT601165
T. williami_05KT601157KT601166
T. costalimaiKC571993KC249327
Triatoma matogrossensisKC249036KC249359
T. matogrossensisKC249038KC249361
Triatoma guazuKC571994KC608984
Triatoma vandaeKC571997KC608989
RubrovariaTriatoma carcavalloiKC248990KC249322
Triatoma circummaculataKC248994KC249323
Triatoma rubrovariaKC249066KC249375
SordidaTriatoma guasayanaKC249342KC249010
Triatoma sordidaKC249077KC249386
T. sordida PAR03KT601158KT601167
T. sordida ITA 24KT601159KT601168
OutgroupPanstrongylus megistusKC248975KC249312

Sequences obtained in this study are given in bold.

Total DNA extraction was performed according to the protocol described by Sambrook and Russel.6 From the extracted DNA, 16S and COI fragments were amplified as described by Sainz and others7 and purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Life Sciences, Buckinghamshire, UK). Purified products were subjected to a sequencing reaction using BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) and were analyzed in the ABI PRISM 377 DNA Sequencer (Applied Biosystems, Warrington, UK). Resulting sequences were edited with BioEdit (version 7.0.5)8 and aligned with ClustalW version 44.9 Nucleotide data for COI were transformed into amino acid sequences to check the alignment for the presence of pseudogenes.10

Combined phylogenetic analyses using both genes were run under a Bayesian framework in MrBayes (version 3.1.45).11 We conducted two independent runs of 1 million generations using four Markov chains and sampling trees every 100 generations (discarding the first 25%). The best evolutionary models were chosen by the Akaike information criterion implemented in the jModelTest (version 0.1.1)12 as follows: for 16S rDNA, HKY + I + G (nst = 2, rates = invgamma) was used; for COI, GTR + I + G (nst = 6, rates = invgamma) was used, yielding a matrix with 22 taxa and 984 nucleotides. Clade support was estimated by Bayesian posterior probabilities (BPP).

The same haplotype was found for all three T. jatai specimens for 16S, on the other hand, none of the three specimens exhibited the same haplotype for COI, but with only five segregating sites. It is in agreement with the faster rate of mutation for COI gene.13 Phylogenetic analysis recovered T. jatai as a sister species to T. costalimai with high clade support (BPP = 98%; Figure 1). Results from both genes were congruent, revealing that T. jatai and T. costalimai exhibit higher p-distances than between valid species. For 16S, the p-distances between T. jatai and T. costalimai were all 0.025. A greater differentiation between T. jatai and T. costalimai was observed for COI (P = 0.097–0.100). For both gene fragments, the distances between T. jatai and T. costalimai were greater than the ones within Rubrovaria (Triatoma rubrovaria, Triatoma circummaculata, and Triatoma caracavalloi) and Infestans subcomplexes (Triatoma infestans, Triatoma platensis, and Triatoma delpontei).

Figure 1.
Figure 1.

Bayesian inference consensus of the combined analysis of sequences of Triatoma species focused on Triatoma jatai based on 16S and COI genes. Molecular evolution models for each partition were HKY + I + G for 16S (444 bp) and GTR + I + G for COI (492 bp). Numbers above the nodes indicate Bayesian posterior probabilities. Panstrongylus megistus was selected as out-group.

Citation: The American Society of Tropical Medicine and Hygiene 94, 3; 10.4269/ajtmh.15-0396

When a taxonomic unit is described, it is important to undertake studies to increase knowledge for the entity in all grounds. Phylogenetic analysis of T. jatai based on mitochondrial genes demonstrated the close genetic relationship between T. jatai and T. costalimai, as it has been initially evidenced by morphological characters and geometric morphometrics of wings.1 Despite the distinctions in taxa set, the topologic position for the remaining species in our tree was similar to the source from where they were downloaded,4,5 as expected.

Evaluating the evolutionary relationships of newly described or sylvatic species is crucial for understanding their vector role in Chagas disease epidemiology.4,5,14 In addition, the taxonomic validity of closely related species is sometimes a matter of questioning,15,16 and molecular approaches must accomplish a good dataset on distinct grounds. The large number of mutations that T. jatai have accumulated in regard to T. costalimai suggests a longer term separation than among members of Infestans subcomplex. Morphological features evaluated for the sympatric T. jatai and T. costalimai1 are supported by our results and confirm the status of T. jatai. What is more, by using 143 field samples of T. jatai, 113 of T. costalimai and other triatomine species, these authors addressed the close relationship between T. jatai and T. costalimai, in agreement with our results.

Mitochondrial genes are easy to sequence and have historically provided great contribution for phylogenetic reconstruction among triatomines.4,10,14 However, Mas-Coma and Bargues13 brought up some limitation for inferences on closely related taxa based on mitochondrial genes, particularly when dealing with sympatric or parapatric species without a clear barrier of isolation. These limitations rely mainly on introgression (sometimes followed by mitochondrial selection), complex population structure, and sex-biased gene flow. Taking into account some weakness of mitochondrial genome for inferences, the further use of nuclear markers (e.g., nuclear ribosomal DNA) is important. It is worth mentioning that females of T. jatai exhibit shorter wings, and are probably unable to fly, as observed by Almeida and others for Triatoma sherlocki,17 also a brachypterous species. The sessile characteristic for females must be taken into account for further population genetic studies.

Recently, Alevi and others18 described the Matogrossensis subcomplex cytogenetically and observed that the species of this subcomplex have the same cytogenetic characteristics as the species of the Rubrovaria subcomplex. Gardim and others10 and Justi and others5 have already brought up the non-monophyly of the Matogrossensis subcomplex. Hence, T. jatai shall be considered for reevaluating this group. Because some clades did not have high clade support within this subcomplex,10 we recommend the use of longer genes, a more complete species set, and multiple molecular markers, also containing nuclear genes. We also strongly recommend studies on the biological cycle, reproductive compatibility between T. jatai and T. costalimai as well as morphological studies of nymphs and antennal phenotype.

  • 1.

    Gonçalves TC, Teves-Neves SC, Santos-Mallet JR, Carbajal-de-la-Fuente AL, Lopes CM, 2013. Triatoma jatai sp. nov. in the state of Tocantins, Brazil (Hemiptera: Reduviidae: Triatominae). Mem Inst Oswaldo Cruz 108: 429437.

    • Search Google Scholar
    • Export Citation
  • 2.

    Obara MT, Barata JMS, da Rosa JA, Almeida PS, Gonçalves GA, Dale C, Gurgel-Gonçalves R, 2012. Description of the female and new records of Triatoma baratai Carcavallo & Jurberg, 2000 (Hemiptera: Heteroptera: Reduviidae: Triatominae) from Mato Grosso do Sul, Brazil, with a key to the species of the Triatoma matogrossensis subcomplex. Zootaxa 3151: 6368.

    • Search Google Scholar
    • Export Citation
  • 3.

    Schofield CJ, Galvão C, 2009. Classification, evolution, and species groups within the Triatominae. Acta Trop 110: 88100.

  • 4.

    Gardim S, Almeida CE, Takiya DM, Oliveira J, Araújo RF, Cicarelli RMB, da Rosa JA, 2014. Multiple mitochondrial genes of some sylvatic Brazilian Triatoma: non-monophyly of the T. brasiliensis subcomplex and the need for a generic revision in the Triatomini. Infect Genet Evol 23: 7479.

    • Search Google Scholar
    • Export Citation
  • 5.

    Justi SA, Russo CA, Mallet JR, Obara MT, Galvão C, 2014. Molecular phylogeny of Triatomini (Hemiptera: Reduviidae: Triatominae). Parasit Vectors 7: 149.

    • Search Google Scholar
    • Export Citation
  • 6.

    Sambrook J, Russell DW, 2001. Molecular Cloning: A Laboratory Manual, 3rd edition. New York, NY: Cold Spring Harbor Laboratory Press.

  • 7.

    Sainz AC, Mauro LV, Moriyama EN, García BA, 2004. Phylogeny of triatomine vectors of Trypanosoma cruzi suggested by mitochondrial DNA sequences. Genetica 121: 229240.

    • Search Google Scholar
    • Export Citation
  • 8.

    Hall TA, 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser 41: 9598.

    • Search Google Scholar
    • Export Citation
  • 9.

    Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG, 2007. Clustal W and Clustal X version 2.0. Bioinformatics 23: 29472948.

    • Search Google Scholar
    • Export Citation
  • 10.

    Gardim S, Rocha CS, Almeida CE, Takiya DM, da Silva MT, Ambrósio DL, Cicarelli RMB, da Rosa JA, 2013. Evolutionary relationships of the Triatoma matogrossensis subcomplex, the endemic Triatoma in central-western Brazil, based on mitochondrial DNA sequences. Am J Trop Med Hyg 89: 766774.

    • Search Google Scholar
    • Export Citation
  • 11.

    Huelsenbeck JP, Ronquist F, 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754755.

  • 12.

    Posada D, 2008. jModelTest: phylogenetic model averaging. Mol Biol Evol 25: 12531256.

  • 13.

    Mas-Coma S, Bargues MD, 2009. Populations, hybrids and the systematic concepts of species and subspecies in Chagas disease triatomine vectors inferred from nuclear ribosomal and mitochondrial DNA. Acta Trop 110: 112136.

    • Search Google Scholar
    • Export Citation
  • 14.

    Hypsa V, Tietz DF, Zrzavý J, Rego RO, Galvao C, Jurberg J, 2002. Phylogeny and biogeography of Triatominae (Hemiptera: Reduviidae): molecular evidence of a New World origin of the Asiatic clade. Mol Phylogenet Evol 23: 447457.

    • Search Google Scholar
    • Export Citation
  • 15.

    Monteiro FA, Costa J, Solé-Cava AM, 1998. Genetic confirmation of the specific status of Triatoma petrochii (Hemiptera: Reduviidae: Triatominae). Ann Trop Med Parasitol 92: 897900.

    • Search Google Scholar
    • Export Citation
  • 16.

    Abad-Franch F, Pavan MG, Jaramillo ON, Palomeque FS, Dale C, Chaverra D, Monteiro FA, 2013. Rhodnius barretti, a new species of Triatominae (Hemiptera: Reduviidae) from western Amazonia. Mem Inst Oswaldo Cruz 108 (Supp 1): 9299.

    • Search Google Scholar
    • Export Citation
  • 17.

    Almeida CE, Oliveira HL, Correia N, Dornak LL, Gumiel M, Neiva VL, Harry M, Mendonça VJ, Costa J, Galvão C, 2012. Dispersion capacity of Triatoma sherlocki, Triatoma juazeirensis and laboratory-bred hybrids. Acta Trop 122: 7179.

    • Search Google Scholar
    • Export Citation
  • 18.

    Alevi KCC, de Oliveira J, Moreira FF, Jurberg J, da Rosa JA, de Azeredo-Oliveira MTV, 2015. Chromosomal characteristics and distribution of constitutive heterochromatin in the Matogrossensis and Rubrovaria subcomplexes. Infect Genet Evol 33: 158162.

    • Search Google Scholar
    • Export Citation

Author Notes

* Address correspondence to Simone Caldas Teves, Laboratório Interdisciplinar de Vigilância Entomológica em Diptera e Hemiptera, Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, Brazil, E-mail: scteves@ioc.fiocruz.br or Carlos Eduardo Almeida, Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas, UNESP, Araraquara, São Paulo, Brazil, and Programa de Pós-Graduação em Ecologia e Monitoramento Ambiental, UFPB, Campus IV, Rio Tinto, PB, Brazil, E-mail: almeida_ce@hotmail.com

Financial support: The study was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brazil) and Fundação de Amparo à Pesquisa do Estado de São Paulo (process nos. 2010/50.355-1, 2010/17027-0, and 2011/22378-0, FAPESP, Brazil).

Disclosure: Ana Laura Carbajal de La Fuente is a member of the “Consejo Nacional de Ciencia y Técnica de la República Argentina” (CONICET) Researcher's Career.

Authors' addresses: Simone Caldas Teves, Laboratório Interdisciplinar de Vigilância Entomológica em Diptera e Hemiptera, Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, Brazil, E-mail: scteves@ioc.fiocruz.br. Sueli Gardim, Departamento de Ciências Biológicas, FCF/UNESP, São Paulo, Brazil, E-mail: sugardim@gmail.com. Ana Laura Carbajal de la Fuente, Laboratorio de Ecoepidemiología, Instituto de Ecología, Genética y Evolución (IEGEBA–CONICET), Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Buenos Aires, Argentina, E-mail: analaura.carbajal@gmail.com. Catarina Macedo Lopes, Teresa Cristina Monte Gonçalves, and Jacenir Reis dos Santos Mallet, Laboratório Interdisciplinar em Vigilância Entomológica em Diptera e Hemiptera, Instituto Oswaldo Cruz/FIOCRUZ, Rio de Janeiro, Brazil, E-mails: aniratac@ioc.fiocruz.br, tcmonte@ioc.fiocruz.br, and jacenir@ioc.fiocruz.br. João Aristeu da Rosa, Departamento de Ciências Biológicas, FCF/UNESP, Sáo Paulo, Brazil, E-mail: rosaja@fcfar.unesp.br. Carlos Eduardo Almeida, Programa de Pós-Graduação em Ecologia e Monitoramento Ambiental, UFPB, Rio Tinto, Paraíba, Brazil, E-mail: almeida_ce@hotmail.com.

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