Phenotypic and genotypic variations among three allopatric populations of Lutzomyia umbratilis, main vector of Leishmania guyanensis
- Moises Thiago de Souza Freitas1,
- Claudia Maria Ríos-Velasquez3,
- César Raimundo Lima CostaJr1,
- Carlos Alberto Santiago FigueirêdoJr1,
- Nádia Consuelo Aragão1,
- Lidiane Gomes da Silva1,
- Marcus Vinicius de Aragão Batista4,
- Teresa Cristina Leal Balbino2,
- Felipe Arley Costa Pessoa3 and
- Valdir de Queiroz Balbino1Email authorView ORCID ID profile
© de Souza Freitas et al. 2015
Received: 18 June 2015
Accepted: 17 August 2015
Published: 4 September 2015
In South America, Lutzomyia umbratilis is the main vector of Leishmania guyanensis, one of the species involved in the transmission of American tegumentary leishmaniasis. In Brazil, L. umbratilis has been recorded in the Amazon region, and in the state of Pernambuco, Northeastern region, where an isolated population has been identified. This study assessed the phylogeographic structure and size and shape differences of the wing of three Brazilian populations.
Samples of L. umbratilis were collected from Rio Preto da Eva (north of the Amazon River, Amazonas), from Manacapuru (south of the Amazon River), and from the isolated population in Recife, Pernambuco state. These samples were processed to obtain sequences of the Cytochrome Oxidase I mitochondrial gene. Geometrics morphometry analysis of the right wing shape of the three populations was made using discriminate canonical analysis.
Phylogenetic analysis revealed the presence of two distinct monophyletic clades: one clade comprised of the Recife and Rio Preto da Eva samples, and the other clade comprised of the Manacapuru samples. Comparing the Manacapuru population with the Recife and Rio Preto da Eva populations generated high indices of interpopulational divergence. Geometric morphometry analysis indicated two distinct groups between the studied populations. Canonical variate analysis of wing shape indicated that Rio Preto da Eva population is significantly closer to Recife population, and both populations were genetically distant from Manacapuru.
The polymorphic sites and geometric morphometry analysis indicate that the distance, lack of continuity and environmental differences have not modified the ancestral relationship between Recife and Rio Preto da Eva populations. The genetic and morphological similarities shared by the Recife and Rio Preto da Eva populations suggest that these populations are more closely related evolutionarily. These results confirm the existence of an L. umbratilis species complex in the North and Northeast regions.
Lutzomyia umbratilis (Diptera: Psychodidae) is the main vector of Leishmania guyanensis, one of the pathogenic agents of American tegumentary leishmaniasis (ATL) [1–3]. This species is found in Brazil, Bolivia, Colombia, French Guyana, Guyana, Peru, Suriname and Venezuela [4, 5]. In Brazil, L. umbratilis is widely distributed in the Amazon basin and there is an isolated population in remnants of the Brazilian Atlantic rain forest in the state of Pernambuco, Northeastern Brazil [4–6]. Of the states in the Northeastern Region, Pernambuco has the third highest number of ATL cases reported in Metropolitan Regions . On the other side of Brazil, in the Amazon region, the presence of L. umbratilis naturally infected with Leishmania guyanensis has been recorded on the east side of the Negro River and the north side of the Amazon River, but not on the south side . In the state of Amazonas, 12,005 cases of ATL were registered between 1994 and 2005; in 2003, 60.18 % of ATL cases occurred in the city of Manaus, where L. umbratilis is recognized as the most significant vector [9, 10].
Considerable geographic distance separates the L. umbratilis populations of the North and Northeast Regions. The low dispersal capacity and geographic isolation of sand flies may result in population structuring, which is amplified by the presence of abiotic barriers [11–13]. The L. umbratilis populations of the North and Northeast Regions are separated by distance and abiotic barriers significant enough to suggest that these individuals may have evolved into genetically differentiated populations.
Bionomical (e.g. fecundity, fertility, and duration of larval development) and genetic differences between L. umbratilis populations on the north and south sides of the Amazon River, suggest that these populations might represent a species complex comprised of at least two incipient species [14, 15]. In light of this possibility, this study aimed to assess the evolutionary relationships between populations of L. umbratilis from the states of Pernambuco and Amazonas.
Field collection and identification of phlebotomine sand flies
Field collections were done in Rio Preto da Eva (north of the Amazon River, Amazonas) (2°50’50”S/59°56’28”W), from Manacapuru (south of the Amazon River) (3°12’41”S/60°26’20) municipalities, located in the Amazonas State, North Region of Brazil, and in the Atlantic Forest Ecological Reserve of Dois Irmãos (8°03’14”S/34°52’52”W) in Recife Municipality, Pernambuco State, Northeast Region of Brazil.
Adult specimens were collected by suction in tree trunks using CDC light traps. The samples were conserved in 95 % alcohol, at -20 °C. The genitalia of the individuals were slide mounted and stored as a voucher, and the sand flies were identified using the keys of Young and Duncan .
DNA extraction, PCR, and sequencing
In all, 201 specimens of L. umbratilis were used in the experiments geometric morphometry and molecular biology. For molecular analysis, 129 specimens of L. umbratilis were used: 36 from Manacapuru (24 males and 12 females); 38 from Rio Preto da Eva (15 males and 23 females); and 55 from Recife (25 males and 30 females).
Genomic DNA extraction was carried out using Chelex®100 (BioRad, Berkeley, California, USA), according to Lima Costa-Junior et al. . A fragment of 597 bp of the Cytochrome Oxidase I (COI) was amplified by PCR using the universal primers CI-J-1632 (+): 5-TGATCAAATTTATAAT-3 and CI-N-2191 (−): 5-GGTAAAATTAAAATATAAACTTC-3, described by Simon et al. . Amplification reactions were done using the Mix Go Taq Colorless kit, according to manufacturer specifications (Promega® Fitchburg, Wisconsin, USA). PCR products were visualized in 1 % agarose gel through UV light and they were purified using the Wizard® SV Gel and PCR Clean-Up System kit (Promega® Fitchburg,Wisconsin, USA). Sequencing was carried out in ABI 3500 automatic sequencer (Applied Biosystems, Cleveland, Ohio, USA). Only sequences with a Phred score above 30 were used in the analysis. Contig assembly was carried out using CodonCode Aligner (CodonCode Corporation). Local alignments were done using BLAST . All new sequences produced in this study have been deposited in GenBank under accession numbers: KM407009 to KM407137.
Nucleotide sequences were aligned using Muscle , incorporated in MEGA v. 5.0 . Phylogenetic analysis was carried out with the Maximum Likelihood criterion using PhyML . The evolutionary model that best fits the data was HKY + G, according to jModelTest . The consistency of the branches was assessed using 1000 bootstrap replicates. Lutzomyia longipalpis, Lutzomyia migonei and Phlebotomus papatasi were used as the outgroup.
Divergence time estimate
The time at which the two main clades diverged (into the Manacapuru and Rio Preto/Recife isolates) was estimated with a relaxed molecular clock using BEAST v. 1.7.5 . The analysis was done for 10,000,000 generations, sampling every 1000 generations. The nucleotide substitution model used was HKY + G. An uncorrelated lognormal clock and a tree prior with Yulebirth process were used. The tree with the higher clade Bayesian Posterior Probabilities (BPP) was obtained with TreeAnnotator v. 1.7.5 (available in BEAST package), using the meanhights option and 10,000 trees as burnin to assure stationarity.
Intra-population genetic diversity was measured by the haplotype and nucleotide diversity, K value (number of genetic groups), number of polymorphic sites, and number of transitions and transversions using DnaSP v. 4.0  and Arlequin v. 3.5 . The frequencies of polymorphic sites were also assessed using WebLogo tool (http://weblogo.berkeley.edu/logo.cgi).
Tajima’s D neutrality test, Fu’s Fs statistics, and the Mismatch distribution were performed using Arlequin v. 3.5 . The Raggedness index (r) and the Sum of squared deviations (SSD) were used in order to validate the model of recent expansion obtained using the Mismatch distribution. Genetic differentiation was assessed with the pairwise fixation index F st using Arlequin v. 3.5 .
The average number of substitutions per site among populations (D xy), the total number of substitutions per site among populations (D a), the number of shared polymorphisms among populations (S s), and the number of fixed differences among populations (S f) were calculated using DnaSP v. 4.0 .
The haplotype network was created with NETWORK v. 4.6 (www.fluxus-engineering.com) using the Median-joining method  to verify the level of haplotype sharing and distribution frequency among the populations.
Genetic structure analysis was performed using Structure v. 2.3 . Interactions were carried out with 20,000 interactions of burning, followed by 200,000 generations of Markov Chain Monte Carlo, adjusted 1 to 10 for each “K” population. Ad hoc quantity ∆K  was used to determine the most accurate number of “K” groups.
Geometric morphometry of the wings
The right wings of adult females of L. umbratilis from the municipalities of Rio Preto da Eva (n = 18), Manacapuru (n = 27) and Recife (n = 27) were dissected with forceps and mounted between microscope slide and cover slide using Berlese fluid. Images of each wing were obtained at 4× magnification using a JVC KY-F55 digital camera coupled to a Leica DM 1000 optical microscope.
The GPA algorithm was used to extract the centroid size; allowing size and shape to be analyzed separately. Size variations were analyzed using the Kruskal-Wallis test. The effect of allometry was analyzed by regression analysis using the coordinates of the landmarks and the centroid size for the three populations. Discriminant analysis of the canonical variables was performed to compare the shape with the overall mean wing size of each population, and the Mahalanobis distances were calculated using 10.000 permutations. To the morphometric statistical analyses we used the softwares TpsUtil 1.60, TpsDig2 v 2.18 (QSC - James Rohlf), TpsRelw 1.54 (QSC - James Rohlf), Tet, MorphoJ 1.06 and Past Program (Paleontological Statistics) v. 2.00.
Intra-population genetic diversity measures for each sample
Rio Preto da Eva (AM)
Genetic differentiation among samples
Manacapuru X Rio Preto da Eva
Manacapuru X Recife
Rio Preto da Eva X Recife
Neutrality tests and population expansion parameters estimated for each sample
Rio Preto da Eva (AM)
The Mismatch distribution also confirms that these populations have recently undergone a process of demographic expansion. The raggedness index (r) did not reject the null hypothesis of recent demographic expansion. The sum of square deviation test (SSD) was not significant. The haplotype fixation index (Fst) was significant (0.89677 – 0.89357) when comparing the Rio Preto da Eva and Recife populations with the Manacapuru population (Table 2)—this reflects a high level of genetic divergence between these two groups.
Analysis indicated the presence of two distinct clades in L. umbratilis: Manacapuru (Clade I), and Rio Preto da Eva and Recife (Clade II). Naturally infected populations of L. umbratilis have been identified in Rio Preto da Eva . The fact that the Recife and Rio Preto da Eva populations are so similar, may suggests that L. guyanensis could be capable of infecting the Recife population. This highlights the necessity of studies that seek to ascertain the vector capacity of the Recife population of L. umbratilis.
The lack of a main natural reservoir (Choloepus didactylus) in the Atlantic forest could act as a limiting factor in the biological cycle of L. guyanensis. Due to the absence of records of this etiological agent in the state of Pernambuco, L. umbratilis has not been incriminated as vectors of ATL despite the high number of cases .
The genetic difference observed between Rio Preto da Eva (north of the Amazon River, Amazonas) and Manacapuru (south of the Amazon River) populations in this study could be related to changes in the course of the Amazon River . The analyzes suggest that changes in the course of the Amazon River initially isolated the L. umbratilis population from Manacapuru for a long period. According to Haffer , separation and a long period of isolation along opposite margins of the river must have had a crucial impact on the speciation of several groups of organisms in the region. This geographic isolation could have caused genetic and bionomic changes in the population from Manacapuru (south of the Amazon River) when compared to the population from Rio Preto da Eva (north of the Amazon River) [14, 15]. This vicariant event happened with other species in this region, such as monkeys and birds .
During this long period of separation among L. umbratilis populations from the central Amazonian, the populations from Rio Preto da Eva (north of the Amazon River) and Recife (Northeast region) remained possibly linked by Amazon and Atlantic forests until the last glacial period. The presence of a clade formed by Rio Preto da Eva and Recife individuals suggests that these populations diverged more recent, possibly during Pleistocene, as occurred in Lutzomyia whitmani . Ready et al.  assessed L. whitmani populations of North and Northeast regions suggesting that there was a continuum of intercrossing between the Amazon and Atlantic forests. This data is supported by our geometric morphometry analysis of wings. These results were unexpected because of the distance, lack of continuity and environmental differences between Amazon and Atlantic forest. The occurrence of several Amazon species of sand flies in Pernambuco reinforce the idea of a continuum between the Amazon and Atlantic forest [36, 37]. However, ecological vicariance should have contributed to the segregation of these populations, primarily via geological and climatic changes—as seen among other organisms in the Amazon region .
Fluctuations of climate and vegetation could have modified the distribution of tropical forests during the Cenozoic, resulting in the retraction and expansion of Amazon vegetation . This process could have contributed to the formation of passages between the Atlantic and Amazon forests. This is a possible explanation for the high level of genetic similarity between the North and Northeast populations. The same scenario was observed in the L. whitmani and L. longipalpis species [39, 40].
In the taxonomic analysis of the L. umbratilis complex, phylogenetic analysis showed that the COI marker used had a high discriminatory capacity; thereby demonstrating the presence of two clades within the Central Amazon and Northeast populations. This gene was used previously to study the L. longipalpis, L. umbratilis, Anopheles albitarsis and Triatoma brasiliensis complexes with considerable efficiency , [41, 42], [43–45]. The applicability of this mitochondrial marker was bolstered by statistical support from the maximum likelihood tree, which detected the presence of two clades for L. umbratilis.
The divergence time estimate indicates that the two clades diverged approximately 0.89 Mya (0.79 – 1.68 Mya), during the Pleistocene. Scarpassa and Alencar  obtained similar results when they analyzed the Northern populations. Therefore, more molecular data will be crucial for establishing a more reliable divergence time estimate.
As with molecular data, GM analysis indicated two distinct groups between the studied populations, with some homogeneity among them. Also, the canonical variate analysis of wing shape indicated that Rio Preto da Eva population is significantly closer to Recife population, and otherwise Manacapuru and Recife populations were more distant. These results indicate divergence between L. umbratilis inhabiting Manacapuru and Recife/Rio Preto da Eva populations. Similar studies using GM and molecular analysis using Lutzomyia shannoni individuals showed divergence between Mexico and USA populations [46, 47].
The markers used in this study (phenotypic and genotypic) allowed the two clades to be differentiated: Manacapuru (Clade I), and Rio Preto da Eva and Recife (Clade II). The genetic similarities shared by the Recife and Rio Preto da Eva populations suggest that Recife individuals could have a vector capacity similar to individuals found north of the Amazon River. However, we cannot be certain that the Recife population possesses vector competence for the transmission of ATL etiological agents. Our evolutionary analysis highlights the necessity for novel studies and for constant surveillance of the L. umbratilis population found in the Atlantic Forest remnant in the state of Pernambuco, Northeastern Brazil.
Ethical approval was not required for the current study.
This study was supported by Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) (APQ-0651-2.02/10), and Universidade Federal de Pernambuco. FACP was supported by PAPES V Program of FIOCRUZ/CNPq, Grant Number 403572/2008-8.
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Lainson R, Ward RD, Shaw JJ. Cutaneous leishmaniasis in North Brazil: Lutzomyia anduzei as a major vector. Trans R Soc Trop Med Hyg. 1976;70:171–2.View ArticlePubMedGoogle Scholar
- Lainson R, Shaw JJ, Ward RD, Ready PD, Naiff RD. Leishmaniasis in Brazil: XIII. Isolation of Leishmania from armadillos (Dasypus novemcinctus), and observation on the epidemiology of cutaneous leishmaniasis in north Pará State. Trans R Soc Trop Med Hyg. 1979;73:239–42.View ArticlePubMedGoogle Scholar
- Lainson R, Shaw JJ, Ward RD, Ready PD, Miles M, Naiff RD. Leishmaniasis in Brazil: XVI. Isolation and identification of Leishmania species from sand flies, wild mammals and man in north Pará State, with particular reference to L. brasiliensis guyanensis causative agent of “pian-bois”. Trans R Soc Trop Med Hyg. 1981;75:530–6.View ArticlePubMedGoogle Scholar
- Young DG, Duncan MA. Guide to the identification and geographic distribution of Lutzomyia sand flies in Mexico, the West Indies, Central and South America (Diptera: Psychodidae). Mem Amer Entomol Inst. 1994;54:881.Google Scholar
- Burgos AM, Hudson JE. Annotated list of the Phlebotominae (Diptera) of Suriname. Mem Inst Oswaldo Cruz. 1994;89:171–8.View ArticleGoogle Scholar
- Balbino VQ, Marcondes CB, Alexander B, Luna LKS, Lucena MMM, Mendes ACS, et al. First report of Lutzomyia (Nyssomyia) umbratilis Ward & Frahia, 1977 outside of Amazonian region, in Recife, State of Pernambuco, Brazil. Mem Inst Oswaldo Cruz. 2001;96:315–7.View ArticlePubMedGoogle Scholar
- 7.Ministério da Saúde: Sistema Nacional de Agravo de notificações. http://www2.datasus.gov.br/DATASUS/index.php. (2013) accessed 12 April 2015.
- Lainson R, Shaw JJ, Silveira FT, Souza AAA, Braga RR, Ishikawa EAY. The dermal leishmaniases of Brazil, with special reference to the eco-epidemiology of the disease in Amazonia. Mem Inst Oswaldo Cruz. 1994;89:435–43.View ArticlePubMedGoogle Scholar
- Guerra JAO, Talhari S, Paes MG, Garrido M, Talhari JM. Aspectos clínicos e diagnósticos da leishmaniose tegumentar americana em militares simultaneamente expostos à infecção na Amazônia. Rev Soc Bras Med Trop. 2003;36:587–90.View ArticleGoogle Scholar
- Guerra JAO, Ribeiro JAS, Coelho LIARC, Barbosa MGV, Paes GP. Epidemiologia da Leishmaniose Tegumentar na comunidade São João, Manaus, Amazonas, Brasil. Cad Saúde Pública. 2006;22:2319–27.View ArticlePubMedGoogle Scholar
- Lanzaro GC, Ostrovska K, Herrero MV, Lawyer PG, Warburg A. Lutzomyia longipalpis is a species complex: genetic divergence and interspecific hybrid sterility among three populations. Am J Trop Med Hyg. 1993;48:839–47.PubMedGoogle Scholar
- Alexander B, Mutebi JP, Hearne D, Lanzaro GC, Ward RD, Hamilton JGC. Current status of the Lutzomyia longipalpis species complex. Mem Inst Oswaldo Cruz. 1998;93:31–3.Google Scholar
- Cohnstaedt LW, Beati L, Caceres AG, Ferro C, Munstermann LE. Phylogenetics of the phlebotomine sand fly group verrucarum (Diptera: Psychodidae: Lutzomyia). Am J Trop Med Hyg. 2011;84:913–22.PubMed CentralView ArticlePubMedGoogle Scholar
- Justiniano SCB, Chagas AC, Pessoa FAC, Queiroz RG. Comparative biology of two populations of Lutzomyia umbratilis (Diptera: Psychodidae) of central Amazonia, Brazil, under laboratory conditions. Braz J Biol. 2004;64:227–35.View ArticlePubMedGoogle Scholar
- 15.Scarpassa VM and Alencar. Lutzomyia umbratilis, the main vector of Leishmania guyanensis, represents a novel species complex? PLoS One. 2012; DOI: 10.1371/journal.pone.0037341.Google Scholar
- 16.Costa-Junior CRL, Freitas MTS, Figueiredo-Junior CAS, Aragao NC, Silva LG, Marcondes CB, Dias Neto RV, Leal-Balbino TC, Souza MBR, Ramalho-Ortigao, M, Balbino VQ. Genetic structuring and fixed polymorphisms in the gene period among natural populations of Lutzomyia longipalpis in Brazil. Parasit Vectors. 2015; doi:10.1186/s13071-015-0785-6Google Scholar
- Simon C, Frati F, Beckenbach A, Crespi B, Liu H, Flook PC. Evolution weighting and phylogenetic utility of mitochondrial gene sequences and compilation of conserved polymerase chain reaction primers. Ann Entomol Soc Am. 1994;87:651–700.View ArticleGoogle Scholar
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.View ArticlePubMedGoogle Scholar
- Edgar RC. Local homology recognition and distance measures in linear time using compressed amino acid alphabets. Nucleic Acids Res. 2004;32:380–5.PubMed CentralView ArticlePubMedGoogle Scholar
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011;28:2731–9.PubMed CentralView ArticlePubMedGoogle Scholar
- Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59:307–2.View ArticlePubMedGoogle Scholar
- Posada D. jmodeltest: phylogenetic model averaging. Mol Biol Evol. 2008;25:1253–6.View ArticlePubMedGoogle Scholar
- Drummond AJ, Suchard MA, Xie D, Rambaut A. Bayesian phylogenetics with BEAUti and the BEAST 1.7.5. Mol Biol Evol. 2012;29:1969–73.PubMed CentralView ArticlePubMedGoogle Scholar
- Rozas J, Sánches-DelBarrio JC, Messeguer X, Rozas R. DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics. 2003;19:2496–7.View ArticlePubMedGoogle Scholar
- Excoffier L, Lischer HEL. Arlequin suite version 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Mol Ecol Resour. 2010;10:564–7.View ArticlePubMedGoogle Scholar
- Bandelt HJ, Forster P, Röhl A. Median-joining networks for inferring intraspecific phylogenies. Mol Biol Evol. 1999;16:37–48.View ArticlePubMedGoogle Scholar
- Pritchard JK, Stephens M, Donnelly P. Inference of population structure using multilocus genotype data. Gen. 2000;155:945–59.Google Scholar
- Evanno G, Regnaut S, Goudet J. Detecting the number of clusters of individuals using the software STRUCTURE: a simulation study. Mol Ecol. 2005;14:2611–20.View ArticlePubMedGoogle Scholar
- Bookstein FL. Morphometric tools for landmark data: geometry and biology. Cambridge: Cambridge University Press; 1991.Google Scholar
- Rohlf FJ, Loy A, Corti M. Morphometric analysis of Old World Talpidae (Mammalia, Insectivora) using partial warp scores. Syst Biol. 1996;45:344–62.View ArticleGoogle Scholar
- Arias JR, Freitas RA. Sobre os vectores da leishmaniose cutânea na Amazônia central do Brasil. 2. Incidência de flagelados em flebotomíneos selváticos. Acta Amaz. 1978;8:387–96.Google Scholar
- Shephard GE, Müller RD, Liu L, Gurnis M. Miocene drainage reversal of the Amazon River driven by plate–mantle interaction. Nat Geosci. 2010;3:870–5.View ArticleGoogle Scholar
- Haffer J. Hypotheses to explain the origin of species in Amazonia. Braz J Biol. 2008;68:917–47.View ArticlePubMedGoogle Scholar
- Gascon C, Williamson GB, Fonseca GAB. Receding forest edges and vanishing reserves. Science. 2000;288:1356–8.View ArticlePubMedGoogle Scholar
- Ready PD, de Souza AA, Macario Rebelo JM, Day JC, Silveira FT, Campbell-Lendrum D, et al. Phylogenetic species and domesticity of Lutzomyia whitmani at the southeast boundary of Amazonian Brazil. Trans R Soc Trop Med Hyg. 1998;92:159–60.View ArticlePubMedGoogle Scholar
- Balbino VQ, Coutinho-Abreu IV, Sonoda IV, Marques SW, Marcondes CB. Phlebotomine sand flies (Diptera: Psychodidae) of the Atlantic forest in Recife, Pernambuco state, Brazil: the species coming to human bait, and their seasonal and monthly variations over a 2-year period. Ann Trop Med Parasitol. 2005;99:683–93.View ArticlePubMedGoogle Scholar
- Dantas-Torres F, Andrade AJ, Tenório KER, Andrade Filho JD, Balbino VQ, Brandão-Filho SP. Phlebotomine sand flies (Diptera: Psychodidae: Phlebotominae) in the State of Pernambuco. Rev Soc Bras Med Trop. 2010;43:733–6.View ArticlePubMedGoogle Scholar
- Haffer J, Prance GT. Impulsos climáticos da evolução na Amazônia durante o Cenozóico: sobre a teoria dos Refúgios da diferenciação biótica. Estud av. 2002;16:175–206.View ArticleGoogle Scholar
- Ishikawa EAY, Ready PD, de Souza AA, Day JC, Rangel EF, Davies CR, et al. A mitochondrial DNA phylogeny indicates close relationships between populations of Lutzomyia whitmani (Diptera: Psychodidae, Phlebotominae) from the rain-forest regions of Amazonia and Northeast Brazil. Mem Inst Oswaldo Cruz. 1999;94:339–45.View ArticlePubMedGoogle Scholar
- 40.Araki AS, Vigoder FM, Bauzer LG, Ferreira GE, Souza NA, Araújo IB, et al. Molecular and behavioral differentiation among Brazilian populations of Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae). PLoS Negl Trop Dis. 2009; doi: 10.1371/journal.pntd.0000365Google Scholar
- Avise JC. Molecular markers, natural history and evolution. New York: Chapman & Hall; 1994.View ArticleGoogle Scholar
- Gomez G, Jaramillo L, Correa MM. Wing Geometric morphometry and molecular assessment of members in the Albitarsis Complex from Colombia. Mol Ecol Resour. 2013;13:1082–92.PubMedGoogle Scholar
- Scarpassa VM, Alencar RB. Molecular taxonomy of the two Leishmania vectors Lutzomyia umbratilis and Lutzomyia anduzei (Diptera: Psychodidae) from the Brazilian Amazon. Parasit Vectors. 2013;6:258.PubMed CentralView ArticlePubMedGoogle Scholar
- Arrivillaga JC, Norris DE, Feliciangeli MD, Lanzaro GC. Phylogeography of the neotropical sand fly Lutzomyia longipalpis inferred from mitochondrial DNA sequences. Inf Genet Evol. 2002;2:83–95.View ArticleGoogle Scholar
- Monteiro FA, Donnelly MJ, Beard CB, Costa J. Nested clade and phylogeographic analyses of the Chagas disease vector Triatoma brasiliensis in Northeast Brazil. Mol Phylogenet Evol. 2004;32:46–56.View ArticlePubMedGoogle Scholar
- Florin DA, Davies SJ, Olsen C, Lawyer P, Lipnick R, Schultz G, et al. Morphometric and molecular analyses of the sand fly species Lutzomyia shannoni (Diptera: Psychodidae: Phlebotominae) collected from seven different geographical areas in the southeastern United States. J Med Entomol. 2011;46:154–66.View ArticleGoogle Scholar
- Florin DA, Rebollar-Téllez EA. Divergence of Lutzomyia (Psathyromyia) shannoni (Diptera: Psychodidae: Phlebotominae) is Indicated by Morphometric and Molecular Analyses when Examined between Taxa from the Southeastern United States and Southern Mexico. J Med Entomol. 2013; doi:org/10.1603/ME13085.Google Scholar