Background Lutzomyia longipalpis is the primary vector of American visceral leishmaniasis. There is strong evidence that L. longipalpis is a species complex, but until recently the existence of sibling species among Brazilian populations was considered a controversial issue. In addition, there is still no consensus regarding the number of species occurring in this complex.Methodology/Principal FindingsUsing period, a gene that controls circadian rhythms and affects interpulse interval periodicity of the male courtship songs in Drosophila melanogaster and close relatives, we analyzed the molecular polymorphism in a number of L. longipalpis samples from different regions in Brazil and compared the results with our previously published data using the same marker. We also studied the male copulation songs and pheromones from some of these populations. The results obtained so far suggest the existence of two main groups of populations in Brazil, one group representing a single species with males producing Burst-type copulation songs and cembrene-1 pheromones; and a second group that is more heterogeneous and probably represents a number of incipient species producing different combinations of Pulse-type songs and pheromones.Conclusions/SignificanceOur results reveal a high level of complexity in the divergence and gene-flow among Brazilian populations of the L. longipalpis species complex. This raises important questions concerning the epidemiological consequences of this incipient speciation process.

The sand fly Lutzomyia longipalpis Lutz & Neiva (Diptera: Psychodidae: Phlebotominae), the main vector of Leishmania infantum in the Americas, is believed to be a species complex, although the status of different Brazilian populations is still somewhat unclear. Preliminary analysis of the acoustic signals that are produced during copulation by L. longipalpis males has suggested the existence of three sibling species in Brazil. In the current report, we analyze in more detail a number of parameters of the copulatory courtship songs of L. longipalpis males from four allopatric populations from different parts of the country (Marajó Island, Natal, Jacobina, and Lapinha Cave) and from two sympatric populations from the locality of Sobral, where two types of males can be differentiated by the number of pale spots (one or two pairs) found on the abdomen. We show that males from the localities of Natal, Marajó, and Sobral (two-spot morph) have very similar songs composed of successive bursts, which are modulated in frequency and amplitude. No significant differences were found in the song parameters of these three populations. In contrast, one-spot males from Sobral and males from Jacobina and Lapinha produce songs that are made of pulses but with distinct patterns for each population and significant differences in all song parameters studied. The results suggest that the L. longipalpis complex in Brazil is composed of four sibling species and that the differences in song patterns between the populations are consistent with the level of divergence found in the period gene.

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Lutzomyia longipalpis s.l. occurs from Argentina to Mexico (Young & Duncan 1994) and because of its wide distribution there have been doubts expressed that this taxon is a single species. Mangabeira (1969) commented on morphological variation seen in males. He observed that specimens captured in the state of Pará, North Brazil, bore a single pair of pale tergal spots on abdominal segment IV (1S males) and those captured from the Northeastern state of Ceará (CE) showed an additional pair of spots on segment III (2S males). Mangabeira commented that the two forms were found in different ecological conditions and speculated that they might represent two species. The existence of a complex was confirmed by Ward et al. (1983Ward et al. ( , 1988 and Lanzaro et al. (1993) in crossing experiments, although the male spot phenotype turned out to be only useful as a morphological marker of different species in some localities such as Sobral, CE (see below) (reviewed in Bauzer et al. 2007).In Brazil, Lutzomyia longipalpis s. l. (Lutz & Neiva, 1912) is without any doubt the principal vector of American visceral leishmaniasis (AVL). It is present in nearly all of the foci of visceral leishmaniasis, is highly anthropophilic as well as readily feeding on dogs and foxes which act as reservoirs of Leishamania chagasi which are frequently found infected (reviewed in Lainson & Rangel 2005).Analysis of male copulatory courtship songs (Souza et al. 2002 and pheromones (Ward et al. 1988, Hamilton et al. 1996a,b, 2005 The best studied populations in Brazil, from an evolutionary point of view, are located in Jacobina (state of Bahía), Lapinha Cave in Lagoa Santa (state of Minas Gerais), Natal (state of Rio Grande do Norte) and Sobral (CE) (Bauzer et al. 2007). The first three localities represent different allopatric siblings (Bauzer et al. 2002b, Bottecchia et al. 2004, Watts et al. 2005) while in Sobral two species coexist in sympatry (Bauzer et al. 2002a, Maingon et al. 2003, Bottecchia et al. 2004. In the present study we compared the reproductive isolation among the three allopatric populations and between the Sobral sympatric siblings.Initially samples of the populations of Jacobina, Lapinha and Natal were obtained using CDC traps (4 to 9 traps in each locality). All males collected in Lapinha had a single pair of spots (1S) in segment IV and all males from Jacobina were 2S (two pairs in segments III and IV, with similar sizes). The majority of males from Natal had the intermediate phenotype, with the extra pair in segment III with less than 3/4 of the size of the spots in segment IV (see Ward et al. 1988). The population from Natal is highly polymorphic for the male spot phenotype but in this locality there is no evidence for the existence of sympatric sibling species (Mukhopadhyay et al. 1998, Bauzer et al. 2002b, Bottecchia et al. 2004, Watts et al. 2005. Eggs from 100 to 200 wild-caught females from each of the three localities were used to establish laboratory colonies following the methodologies 217 Reproductive isolation...

BackgroundBrazilian populations of Lutzomyia longipalpis may constitute a complex of cryptic species, and this report investigates the distribution and number of potential sibling species. One of the main differences observed among Brazilian populations is the type of acoustic signal produced by males during copulation. These copulation song differences seem to be evolving faster than neutral molecular markers and have been suggested to contribute to insemination failure observed in crosses between these sibling species. In previous studies, two main types of copulation songs were found, burst-type and pulse-type. The latter type can, in turn, be further subdivided into five different patterns.MethodsWe recorded male song from 13 new populations of the L. longipalpis complex from Brazil and compared the songs with 12 already available.ResultsOut of these 25 populations, 16 produce burst-type and 9 produce pulse-type songs. We performed a principal component analysis in these two main groups separately and an additional discriminant analysis in the pulse-type group. The pulse-type populations showed a clear separation between the five known patterns with a high correspondence of individuals to their correct group, confirming the differentiation between them. The distinctiveness of the burst-type subgroups was much lower than that observed among the pulse-type groups and no clear population structure was observed. This suggests that the burst-type populations represent a single species.ConclusionOverall, our results are consistent with the existence in Brazil of at least six species of the L. longipalpis complex, one with a wide distribution comprising all the populations with burst-type songs, and five more closely related allopatric siblings with different pulse-type song patterns and more restricted distribution ranges.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0900-8) contains supplementary material, which is available to authorized users.