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    A, T. sherlocki; B, T. brasiliensis; C, T. melanica; D, T. juazeirensis.

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    Brazil northeastern region with distribution of T. brasiliensis complex according to Costa et al. (2002). This figure appears in color at www.ajtmh.org.

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    Neighbor-joining tree based on Kimura 2-parameter distances among members of T. brasiliensis complex, with mtCytB and 16S. (A) phylogram resulting from 399 bp sequence alignment of mtCytB; (B) phylogram resulting from 316 bp sequence alignment of 16S mitochondrial ribosomal RNA. The values of the nodes indicate the percentage of the posterior probability. T. sordida and T. infestans were used as outgroups.

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    Tree derived from maximum likelihood (ML) estimation by Bayesian inference applied to fragments from CytB and 16S. The values over the nodes refer to Kimura 2-parameter distances. Those below the nodes indicate the percentage of clade credibility by Bayesian analysis, while those beside the nodes are the bootstrap value by maximum parsimony.

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Phylogeny of Triatoma sherlocki (Hemiptera: Reduviidae: Triatominae) Inferred from Two Mitochondrial Genes Suggests Its Location Within the Triatoma brasiliensis Complex

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  • 1 Departamento de Parasitologia, Instituto de Biologia–UNICAMP, Campinas, SP, Brazil; Departamento de Ciências Biológicas, Faculdade de Ciências Farmacêuticas–UNESP Araraquara, SP, Brazil; Secretaria de Saúde do Estado da Bahia, SESAB, Salvador, BA, Brazil; Centro de Estudos de Insetos Sociais, Instituto de Biociências–UNESP Rio Claro, SP, Brazil; Laboratório de Biodiversidade Entomológica, Instituto Oswaldo Cruz–Fiocruz, Rio de Janeiro, RJ, Brazil; Departamento de Análises Clínicas, Faculdade de Ciências Farmacêuticas–UNESP Araraquara, SP, Brazil

The phylogenetic position of Triatoma sherlocki within triatomines group was inferred by analyzing mtDNA fragments of Cyt B and 16S ribosomal RNA by using maximum parsimony and Bayesian analysis. Despite being differentiated from members of the T. brasiliensis complex on morphologic grounds, molecular phylogenetic analysis suggests T. sherlocki is a member of this complex; moreover, it was placed as a sister species of T. melanica. These suggestions were supported by robust credibility rates. Hence, we show evidence for the paraphyletic group of the “Triatoma brasiliensis complex,” which should be composed of T. brasiliensis brasiliensis, T. brasiliensis macromelasoma, T. juazeirensis, T. melanica, and T. sherlocki.

INTRODUCTION

The great importance of the Triatominae, a subfamily of blood-sucking bugs, in public health terms, derives from its capacity to transmit Trypanosoma cruzi, the etiologic agent of Chagas disease.1 The overall prevalence of human infection is estimated at 15 million cases. Approximately 28 million inhabitants of Latin America are at risk of contracting this infection.2

Vector transmission is still considered the main route of human infection; therefore, most of the effort to prevent Chagas disease turns on the interruption of this transmission by controlling domestic vectors with pyrethroid insecticides. The Brazilian National Health Foundation (FUNASA) has used intense insecticide spraying since the 1990s in Brazil. An important achievement of this program has been the interruption of T. cruzi transmission by Triatoma infestans.2,3

The last checklist of triatomines included 137 valid species, distributed in 19 genera and 6 tribes.4 However, these numbers do not reflect the actual scenario. Continuous changes take place in light of taxonomic reviews, and new species are described. At the time of writing 143 species and 18 genera have been recognized.512

Triatoma sherlocki Papa and others, 2002 is a highly endemic and wild species, described from specimens collected in the Santo Inácio district, of the town of Gentio do Ouro, Bahia state, Brazil. 13 This species was formerly recorded as a subspecies of T. brasiliensis Neiva, 1911 (T. brasiliensis santinacensis, Rui Cerqueira; unpublished data).

After the successful vector control program for T. infestans, T. brasiliensis became the main vectorial threat in semi-arid areas of northeastern Brazil. 2,4,14

Triatoma brasiliensis shows remarkable chromatic variation across its geographic distribution. 15 This characteristic led this “species” to be subjected to a multi-disciplinary study of its morphology, biology, ecology, isoenzymes, and mitochondrial DNA variation. 14,1620 It was concluded that T. brasiliensis is in fact a complex of species based on three allopatric evolutionary units: T. brasiliensis Neiva, 1911, T. brasiliensis melanica Neiva and Lent, 1941 and T. brasiliensis macromelasoma Galvão, 1956. 15

Molecular markers based on DNA have led to considerable progress in the understanding of genetic relatedness, phylogeny, and population dynamics in triatomines during the last 10 years. 2124 However, these studies have not included rare, wild, or newly described species. Because T. sherlocki is a newly described species, the aim of this study is to clarify the consistency of the morphologic information used to suggest that T. sherlocki is a species closely related to T. brasiliensis.

MATERIALS AND METHODS

Insects.

Triatoma sherlocki (Figure 1A) samples used in this study were taken from a colony maintained at the Triatominae Insectarium of the São Paulo State University (UNESP, Araraquara, Brazil). The colony was generated from 26 specimens collected in a wild and rock upland environment, near the village of Santo Inácio (11°06′55″ S and 42°46′30″ W) in the district of Gentio do Ouro, state of Bahia, Brazil, in 2003 (Figure 2).

Triatoma brasiliensis (Figure 1B) specimens used were from a colony founded from 52 specimens collected in rural houses in São João do Piauí, State of Piauí (8°21′29″ S and 42°14′48″W) in 1984 and maintained since then at the same Insectarium.

Triatomamelanica (Figure 1C) specimens were obtained from a colony maintained at Instituto Oswaldo Cruz (FIOCRUZ). The founders of this last colony were collected in the district of Espinosa (14°55′34″ S and 42°49′09″ W), a town in the state of Minas Gerais, among rock piles in a rocky wildness.

DNA extraction, PCR amplification, cloning, and sequencing.

All six legs from one specimen of each species were cut with a sterile needle and ground in a microcentrifuge tube with a clean crusher. The total genomic DNA was extracted from single specimens by the standard phenol/chloroform technique and stored at −4°C until further use. 25

Two target mitochondrial DNA fragments were amplified by polymerase chain reaction (PCR), resulting in a 414-bp fragment of the Cyt B gene and a 373-bp fragment of the 16S large subunit ribosomal RNA region of the mitochondrial gene. The DNA fragments were amplified with the following primer combinations: Cyt B (F: 5′-GGA CAA ATA TCA TTT TGA GGA GCA ACA G-3′) and (R: 5′-ATT ACT CCT CCT AGC TTA TTA GGA ATT G-3′) and 16S mitochondrial gene (F: 5′-CRC CTG TTT AAC AAA AAC AT-3′ and R: 5′-AAA AAA ATT ACG CTG TTA TCC CTA AAG TAA-3′), performed in a PTC–100 thermal cycler (MJ Research). 21 The following reactions conditions were used: denaturation at 94°C for 5 minutes and then 35 cycles of denaturation at 94°C for 45 seconds, annealing at 50°C for 45 seconds, and extension at 72°C for 1 minute, followed by a 72°C extension for 7 minutes and storage at 4°C indefinitely, for the both gene fragments.

The resulting amplicons were separated on a 1.5% agarose gel, and DNA fragments were extracted from the gel with an Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare Life Sciences, Upsala, Sweden), following the manufacturer’s instructions, cloned into the pGEM-T vector (Promega, Madison, WI), and sequenced in an ABI PRISM 377 DNA Sequencer (Perkin Elmer, Wellesley, MA), using the Big Dye Terminator v1.0 Cycle Sequencing kit (Applied Biosystems, Foster City, CA).

Phylogenetic analysis.

Sequences of DNA generated in this study, as well as other sequences from the GenBank, were edited with the program BioEdit 7.0.5 and aligned with the program ClustalW, using default parameters. 26,27 Because indels are common for the 16S fragment, a pilot test was performed including more species in the alignment. However, no region of ambiguity was detected, and the pilot tree based on maximum parsimony (MP) analysis exhibited similar profile already shown by Garcia and Powell 28 and Garcia and others. 22

The alignments were cropped in accordance with the shortest sequence and analyzed for Kimura 2-parameter distance producing neighbor-joining and phylogenetic trees through the software MEGA 3.0, whereas MP analysis was conducted using PAUP 4.0b10. 29,30 Clade support was estimated by 1,000 replications for both analyses. MP analyses were used with TBR (tree-bisection-reconnection) branch swapping. The TBR is the default search strategy in both PAUP* and MrBayes. It defines the branch lengths, being largely applied and exhibiting a satisfactory degree of reliability of branch lengths.

The choices of taxa to build the trees were made according to availability in the GenBank and to each question. We also used information provided by other authors to choose the most related taxa and the outgroups. 21,22,28

The phylogeny was also analyzed by Bayesian inference with independent runs of Markov chains. Clade support was estimated using a Markov chain Monte Carlo (MCMC) algorithm performed by the program MrBayes version 3.1. 31 The first analysis was run for 1,000,000 generations, with sampling every 250 generations; it continued running until establishing the average SD of split frequencies. We used estimates of likelihood settings by using MrModeltest and chose the Akaike information criterion (AIC) 32 to obtain the best model for each gene and their posterior probability of credibility. The AIC indicates the best model to be applied to a given gene fragment (16S and Cyt B, in our case), by measuring of the goodness of fit of all possible statistical models. 33

Thus, we used different models for the different partitions indicated by AIC. For Cyt B, we used the general model of DNA substitution with gamma-distributed rate variation across sites (GTR + G), assuming that all six substitution types have separate rate ratios (nst = 6), and three-codon partition. For the 16S fragment, we used the GTR model with two substitution types that only distinguishes between transitions and transversions (nst = 2), a proportion of sites being invariant (rates = propinv). The default random tree option was used to begin the analysis. Log-likelihood values from four simultaneous MCMC chains stabilized at 8,000,000 generations, where the average SD of split frequencies was 0.004. Summarizing the sampled parameter values by using the sump command, we observed that trees sampled during the first 2,000,000 generations of the run did not converge onto a stationary point, showing very different trees. Therefore, those first trees were discarded from the analysis, corresponding to 25% of our samples to avoid having a negative influence in the posterior probability values. Triatoma infestans and T. sordida were used to root the trees.

RESULTS

Cytochrome b (Cyt B).

The amplified fragment of the Cyt B region produced a sequence with 414 bp for both T. sherlocki and T. brasiliensis. The alignment was performed with the following species: T. sherlocki, T. brasiliensis, T. brasiliensis brasiliensis, T. brasiliensis macromelasoma, T. juazeirensis, T. melanica H, T. infestans, and T. sordida. The resulting consensus sequence had 399 bp, being shortened to the length of the T. sordida sequence. This alignment exhibited 120 (30%) polymorphic sites and 132 substitutions (30 transversions and 102 transitions). GenBank accession numbers and references with details about the species are shown in Table 1. Third-base modifications corresponded to 77% of the 120 polymorphic sites, resulting in 12 non-synonymous substitutions (10%) among the set of species chosen (Table 2).

The neighbor-joining tree built with the set of taxa cited above, based on the Kimura 2-parameter distance model, showed that T. sherlocki was placed as the sister species of the newly described T. melanica (Figure 3A). The robustness of credibility rates was increased when T. sordida was taken out of the analysis (all > 73), permitting analysis of a fragment of 414 bp. The uncorrected p-distances between T. sherlocki and the two haplotypes of T. melanica were 0.085 and 0.094. These distances were not appreciably lower than those between T. sherlocki and T. juazeirensis (0.094–0.114).

The phylogenetic closeness between T. sherlocki and T. melanica was also observed from the genetic translation of the sequences of the species involved. The sequence of 133 amino acids derived for the Cyt B DNA sequence exhibited a polymorphism in only one amino acid between these two species.

The Cyt B haplotype that we obtained here for T. brasiliensis from Ceará was the same one designated by Monteiro and others 20 as haplotype “v,” agreeing with the subspecific taxonomic characteristics of T. brasiliensis brasiliensis and with the geographic distribution of this taxon.

Bayesian analysis with this gene showed strong support for the allocation of T. sherlocki inside the T. basiliensis complex (posterior probability [pp] = 99) and for its placement as a sister species to T. melanica (pp = 97%).

Large subunit ribosomal RNA (16S).

The amplification of the mtDNA for 16S rRNA generated a fragment with 373 bp for T. sherlocki and T. melanica, and 374 bp for T. brasiliensis. After aligning our sequences with the ones from T. infestans and T. sordida, we had a consensus sequence of 316 bp. This alignment exhibited 45 substitutions, of which 28 were transitions, 17 were transversions, and 41 were polymorphic sites (13%; Table 3). Accession numbers codes and references with details about the species are shown in Table 1.

A p-distance tree was built using the Kimura 2-parameter model, where T. sherlocki and T. melanica were again placed as sister species, with a low bootstrap value (71). However, for this gene, T. sherlocki was also relatively well supported (bootstrap = 81) as a member of the T. brasiliensis complex (Figure 3B). These fragment provided few informative sites, and further analysis (MP and Bayesian) did not increase the robustness of credibility for this gene alone. As observed for Cyt B, T. sordida and T. infestans were the outer species, T. infestans being a little closer (p-distances) to the T. brasiliensis complex (Figure 3B).

Bayesian analysis with Cyt B and concatenated data.

Only Cyt B was considerably informative considering our question, and the Bayesian tree built this genes (Cyt b and 16S) and the representative members of T. brasiliensis complex placed T. sherlocki as a sister to T. melanica ( Figure 4 ) with 97% of posterior probability.

The incongruence length difference (ILD) test allowed the two mtDNA fragments studied here to be combined. 34 The close relationship between T. sherlocki and the members of T. brasiliensis complex observed in previous results was confirmed with a very slight robustness for MP analysis. However, for Bayesian analysis, we observed 100% of credibility to support this branch (Figure 4).

DISCUSSION

Triatoma sherlocki (Figure 1A) is a sylvatic species collected and studied for the first time by Cerqueira (1982) (unpublished data), being originally considered a subspecies of T. brasiliensis: T. brasiliensis santinacensis. The specimens used for its description were collected ~250 m from a small town (Gentio do Ouro), district of Santo Inácio, in the Chapada Diamantina range, an extended plateau rich in rock formations that rises from 400 to 2,000 m above sea level in the central region of Bahia state. Since its description, it has not been found in any other part of northeastern Brazil (Figure 2). According to Cerqueira (1982) (unpublished data), the habitats of T. sherlocki are rocky ecotypes without vegetation, around an elevation of 470 m above sea level, where sylvatic lizards and rodents (Trichomys cunicularius, Kerodon rupestris) are common. This species has peculiar characteristics, such as shorter fore and hind wings (brachypterous) and red-orange spots or stripes in the connexivum and legs, respectively (Figure 1A). 13

Triatoma brasiliensis (Figure 1B) was described from specimens collected in Caicó, Rio Grande do Norte state, in northeastern Brazil. 35 Lent and Wygodzinsky 15 considered T. brasiliensis melanica Neiva and Lent, 1941 and T. brasiliensis macromelasoma Galvão, 1956, as belonging to the same species as T. brasiliensis. Costa35 reported that T. brasiliensis was in fact composed of four distinct populations: brasiliensis (Caicó, RN), melanica (Espinosa, MG), macromelasoma (Petrolina, PE), and juazeirensis (Juazeiro, BA). Costa and others,7 through morphologic and enzymatic studies, concluded that T. melanica (Figure 1C) was a new species. Based on the same approaches, 1 year later, Costa and Felix 10 described another species, T. juazeirensis (Figure 1D), which had been previously considered only a population of T. brasiliensis complex.

This study presents data suggesting that T. sherlocki is a member of the T. brasiliensis complex by sequencing two distinct mitochondrial genes (Figure 4). In addition, the placement of T. sherlocki within the T. brasiliensis complex suggests that it is a sister species of T. melanica, with strong clade support (98% credibility; Figure 4).

A profile of rates of polymorphic sites observed for other taxa for the ribosomal 16S and Cyt B mtDNA fragments was found here (Tables 2 and 3). Although the 16S sequence showed polymorphic sites in only 13% (41/316; Table 2) of aligned base pairs, the Cyt B gene showed 30% (120/399; Table 1) of polymorphic sites among the following species set: T. sherlocki, T. brasiliensis brasiliensis, T. brasiliensis macromelasoma, T. melanica, T. juazeirensis, T. infestans, and T. sordida (Table 3).

Despite being originally considered a subspecies of T. brasiliensis based on laboratory crossing experiments (Cerqueira 1982, unpublished data). Posteriorly Papa and others 13 described T. sherlocki that presented morphologic distinguishable features of T. lenti, suggesting that T. sherlocki could be a closely related species. Therefore, considering the unusual morphologic features of T. sherlocki, it was not expected to be placed within the T. brasiliensis complex, where the members differ among them mainly by a few details in the chromatic pattern (Figure 1). For that reason, T. melanica was classified as new taxa considering a peculiar characteristic in male genital: a spongy fossula on the fore tibia.7 Despite the marked variation in male genital structures among the members of the T. brasiliensis complex, this feature by itself was not useful to distinguish these species.7 On the other hand, T. sherlocki can easily be differentiated from all members of the T. brasiliensis complex by genital structures, the particular red-orange details, and very small wings (Figure 1A).

Disagreements between morphologic and genetic approaches have also been recorded for other taxa among the triatomines, such as the paraphyletic group of Triatoma genera that should include some species of Panstrongylus genera and the discussion about Psammolestes genera as a closely related genera to Rhodnius, despite being very different morphologically (both originally evidenced by Hypsa and others). 36

All members of the T. brasiliensis complex are found in rock piles within the biogeographic zone known as the Caatinga, a mosaic of xerophytic, deciduous, semi-arid thorn scrubs and forest. 37 However, an ecologic niche model for the T. brasiliensis complex showed that each species of this complex occupies an environment with particular features, which might have driven the specialization and, therefore, the genetic drift. 38 Despite apparently being physically closer to the geographic area occupied by T. juazeirensis (Figure 1D), T. sherlocki is genetically closer to T. melanica. However, it is necessary point out that the real geographic distribution of T. sherlocki was poorly studied, because it was described in a remote region in the center northwestern part of the state of Bahia, Brazil, and its presence was restricted to a very small area. Along this region, there is a gap between the known geographic distributions of the two species of the T. brasiliensis complex that occur in this state: T. melanica and T. juazeirensis. The former is restricted to the southern part of the Brazilian semi-arid area, whereas the latter occupies the middle part, south of São Francisco River, and T. brasiliensis is found in the region to the north of São Francisco River.

Among the four members of the T. brasiliensis complex, the subspecies T. brasiliensis brasiliensis has been considered the main concern in terms of Chagas disease transmission, because this subspecies is the most widespread and shows the highest rates of domestic capture and of natural infection by T. cruzi. On the other hand, T. melanica has been found exclusively in wild habitats thus far, whereas T. brasiliensis brasiliensis, T. brasiliensis macromelasoma, and T. juazeirensis can be found in both wild and domestic environments. 14 T. sherlocki seems to be a sylvatic species, but it eventually invades homes in Santo Inácio (FUNASA, personal communication).

However, the epidemiologic profile of Chagas disease vectors is not static. Some wild vectors are progressively coming closer to human habitation, in the process of domiciliation. For example, since the control of T. infestans in the south of Brazil, data from the Chagas Disease Control Program have indicated an increase in the invasion of housing and nearby areas by T. rubrovaria, which is becoming the most frequently captured triatomine species in that region. 3941 A similar pattern has been observed for R. nasutus in the state of Ceará, where T. brasiliensis, R. nasutus, and T. pseudomaculata have been frequently captured inside houses. 42,43 Additional studies are needed on T. sherlocki, by analyzing other genes, to confirm its phylogenetic position. In addition, it is suggested that the knowledge of this species must be strengthened through morphologic, biologic, ecologic, and biogeographic studies, which could provide valuable information on its taxonomic position and vector capacity.

From our results, the close phylogenetic relationship between T. sherlocki and the species of the T. brasiliensis complex, especially with the T. melanica, inferred from Bayesian trees (Figures 3 and 4) support the hypothesis that T. sherlocki can be classified as one specie of the T. brasiliensis complex and might indicate possible philogeography from a common ancestry.

Table 1

Accession numbers and references with details about the species available at GenBank

Table 1
Table 2

Variable sites in 399 bp of the mitochondrial Cyt B gene for eight species of bugs

Table 2
Table 3

Variable sites in 316 bp of the mitochondrial 16S gene for five bugs

Table 3
Figure 1.
Figure 1.

A, T. sherlocki; B, T. brasiliensis; C, T. melanica; D, T. juazeirensis.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 5; 10.4269/ajtmh.2009.08-0664

Figure 2.
Figure 2.

Brazil northeastern region with distribution of T. brasiliensis complex according to Costa et al. (2002). This figure appears in color at www.ajtmh.org.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 5; 10.4269/ajtmh.2009.08-0664

Figure 3.
Figure 3.

Neighbor-joining tree based on Kimura 2-parameter distances among members of T. brasiliensis complex, with mtCytB and 16S. (A) phylogram resulting from 399 bp sequence alignment of mtCytB; (B) phylogram resulting from 316 bp sequence alignment of 16S mitochondrial ribosomal RNA. The values of the nodes indicate the percentage of the posterior probability. T. sordida and T. infestans were used as outgroups.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 5; 10.4269/ajtmh.2009.08-0664

Figure 4.
Figure 4.

Tree derived from maximum likelihood (ML) estimation by Bayesian inference applied to fragments from CytB and 16S. The values over the nodes refer to Kimura 2-parameter distances. Those below the nodes indicate the percentage of clade credibility by Bayesian analysis, while those beside the nodes are the bootstrap value by maximum parsimony.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 81, 5; 10.4269/ajtmh.2009.08-0664

*

Address correspondence to João A. da Rosa, Rod. Araraquara-Jaú, Km 1, Faculdade de Ciências Farmacêuticas/UNESP Araraquara, Departamento de Ciências Biológicas, Laboratório de Parasitologia, CEP 14801-902 Araraquara/SP, Brazil. E-mail: rosaja@fcfar.unesp.br

Authors’ addresses: Vagner J. Mendonça, Marco T. A. da Silva, Márcia A. S. Graminha, Regina M. B. Cicarelli, and João A. da Rosa, Rod. Araraquara-Jaú, Km 1, Faculdade de Ciências Farmacêuticas, Universidade Estadual Paulista, CEP 14801-902, Araraquara, São Paulo, Brazil, Tel: 55-16-33016943, E-mails: v041137@dac.unicamp.br, tulio@fcfar.unesp.br, graminha@fcfar.unesp.br, cicarell@fcfar.unesp.br, and rosaja@fcfar.unesp.br. Renato F. de Araújo, Secretaria de Saúde do Estado da Bahia, CEP 41750-300, Salvador, Bahia, Brazil, Tel: 55-71-32705821, E-mail: birdeagle01@yahoo.com.br. Joaquim Martins Júnior and Maurício Bacci Júnior, Avenida 24-A, 1515, Bela Vista, Centro de Estudos de Insetos Sociais, Universidade Estadual Paulista, CEP 13.506-900, Rio Claro, São Paulo, Brazil, Tel: 55-19-35264160, E-mails: redskull_br@yahoo.com.br and mbacci@rc.unesp.br. Carlos Eduardo Almeida and Jane Costa, Instituto Oswaldo Cruz, Fiocruz, Pavilhão Mourisco, Room 202, Av. Brasil 4365, 21045-900 Rio de Janeiro, RJ, Brazil, Fax: 55-21-2573-7276, E-mails: almeidace@ioc.fiocruz.br and jcosta@ioc.fiocruz.br.

Acknowledgments: The authors thank João Luis Molina Gil and João Mauricio Nóbrega da Silva Filho who maintained the Triatominae colonies, Rui Cerqueira who pioneered the collection and study of T. sherlocki, José Clóvis for critical suggestions, and Timothy J. C. Roberts for suggestions and proofreading of the English text.

Financial support: This work was supported by Fundação para o Desenvolvimento da UNESP (FUNDUNESP, Process 066/06) and Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP, Process 06/02778-5 and 05/52608-6).

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