*Departamento de Bioquímica e Biologia Molecular, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil;
Departamento de Entomologia, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil;
The Centre for Applied Entomology and Parasitology, School of Life Sciences, Keele University, Keele, UK;
Department of Genetics, University of Leicester, Leicester, UK
There is mounting evidence that the sandfly Lutzomyia longipalpis (Diptera: Psychodidae: Phlebotominae) (Lutz and Neiva 1912), the main vector of the visceral leishmaniasis in Latin America, is a complex of sibling species (Lanzaro et al. 1993
; Dujardin et al. 1997
; Lampo et al. 1999
; Uribe 1999
; Arrivillaga and Feliciangeli 2001
; Soto et al. 2001
). But, the evidence for Brazilian populations is still contradictory. Crossing experiments, pheromonal (Ward et al. 1983
, 1988
; Hamilton et al. 1999a
, 1999b
), copulation songs (Souza et al. 2002
) and molecular analyses (Bauzer et al. 2002
) all support the idea of a species complex and seem to confirm the data obtained for other populations of this species. But work with isoenzyme loci suggests that although it is a highly polymorphic and geographically variable species, L. longipalpis should not be considered a species complex in Brazil (Mukhopadhyay et al. 1998b
; Mutebi et al. 1999
; Azevedo et al. 2000
).
Observation on morphological variation in tergal spot patterns was the first indication that L. longipalpis could be a species complex (Mangabeira 1969
). Specimens bearing only a single pair of pale spots on the fourth tergite were shown to occur from Mexico to southern Brazil, whereas males bearing an additional pair of spots on the third tergite were shown to be more concentrated in Northeastern Brazil, although they were also found in other regions (Ward et al. 1985
, 1988
). Crosses between one- and two-spot populations in some cases produced an intermediate phenotype, a small spot on the third tergite (Ward et al. 1988
), and in some populations, mainly along the Northeastern coast, one- and two-spot males are found together with high frequencies of intermediate forms, indicating the existence of an intraspecific polymorphism in those areas (Ward et al. 1988
; Mukhopadhyay et al. 1998a
).
In contrast, there are also some localities in Brazil such as Sobral (Ceará State) where the two forms occur sympatrically with the near absence or very reduced number of intermediate forms (Ward et al. 1988
; Mutebi et al. 1999
). Reproductive isolation in laboratory crosses and pheromone differences between one- and two-spot sandflies from this locality strongly suggest that this absence of intermediate forms reflects the coexistence of two sibling species (Ward et al. 1988
). But the fact that isoenzyme genotypic frequencies in Sobral and other populations with small numbers of intermediate forms were usually in accordance with Hardy-Weinberg expectations (Mutebi et al. 1999
) was regarded as evidence against the sympatric sibling species hypothesis. It was hypothesized, but not tested, that the lack of intermediates in these populations could be caused by a shift from semidominance to dominance in the genetic basis of the spot phenotype, caused perhaps by an environmental effect (Mutebi et al. 1999
). Therefore, the issue concerning the existence of a species complex in Brazil remains contentious.
The period (per) is a Drosophila clock gene (Konopka and Benzer 1971
) that has been implicated in the control of species-specific differences in locomotor and mating activity (Petersen, Hall, and Rosbash 1988
; Sakai and Ishida 2001
) and in the lovesong that males produce during courtship (Kyriacou and Hall 1980
, 1982
, 1986
; Wheeler et al. 1991
; Ritchie, Halsey, and Gleason 1999
). These behavioral phenotypes are potentially important in maintaining the reproductive isolation between closely related species, thus per has been called a speciation gene (Coyne 1992
) and has been used as a molecular marker in a number of studies of closely related species of Drosophila (reviewed in Peixoto 2002
). Therefore, even though the role of per in the speciation of sandflies has not yet been demonstrated, it is a relevant gene for such kinds of studies.
Recently, we used a 266-bp fragment of the L. longipalpis per gene homologue as a molecular marker to study the differentiation between three allopatric populations from Brazil (Lapinha, Jacobina and Natal, see map in the Supplementary Material at the MBE website: www.molbiolevol.org), and our results indicated a high level of differentiation among them (Fst = 0.4661 and Nm = 0.2863, both values calculated for the three populations together; see Bauzer et al. 2002
). In the current report we used the same region of per to analyze the genetic differentiation between one- and two-spot males from the locality of Sobral, Ceará state, Brazil (3°41'S, 40°20'W) in an attempt to determine if they represent two different sibling species. We analyzed sequences from 24 one-spot males (Sobral 1S) and 23 two-spot males (Sobral 2S) collected in November 1999 using primers 5Llper1 (5'-CAATGGCTTCTACATCACTC-3'), 3Llper1 (5'-ACTTGCTGCTTCACTGTATC-3'), and the methods described in Bauzer et al. (2002)
. Analyses were performed using DnaSP3.5 (Rozas and Rozas 1999
), ProSeq (Filatov and Charlesworth 1999
), and MEGA 2.1 (Kumar et al. 2001
). The sequences have been submitted to GenBank (accession numbers AY082911AY082957), and an alignment is available as Supplementary Material.
All the observed differences between the 47 sequences were either synonymous changes or they occurred within the intron. Estimates of the nucleotide diversity (), number of segregating sites (S), and neutral parameter (
) show that Sobral 1S (
= 0.0243, S = 27, and
= 0.0272) is more polymorphic than Sobral 2S (
= 0.0173; S = 22 and
= 0.0224). In fact, Sobral 1S is the most polymorphic population found so far with respect to per, as
varied from 0.0148 to 0.0209 and
ranged from 0.0188 to 0.0206 in the populations of Lapinha (23 sequences), Jacobina (22), and Natal (21) (see Bauzer et al. 2002
). We applied Tajima's (1989)
and Fu and Li's (1993) tests of natural selection, and in both cases the D values were not significant, indicating no obvious departures from neutrality (Sobral 1S: DTajima = -0.417 and DFu&Li = -0.322; Sobral 2S: DTajima = -0.852 and DFu&Li = -0.979).
Table 1
shows the pairwise fixation index (Fst) and the estimated number of migrants per generation (Nm) between Sobral 1S and 2S that indicate the degree of differentiation between the two populations (Hudson, Slatkin, and Maddison 1992
). The two groups are clearly distinct with a Fst value of 0.3952 and only 0.3827 migrants per generation. Figure 1
shows a neighbor-joining tree (Saitou and Nei 1987
) with all alleles of both forms constructed using Kimura's two parameters distance. Lutzomyia dispar (accession number AY071912), another member of the Lutzomyia subgenus, was used as an outgroup to root the tree. Even though the majority of bootstrap values are below 50%, probably because of the high number of sequences and the small number of informative sites, we can see that, except for one sequence (2S24br1027), all other alleles of Sobral 2S form a monophyletic group.
|
|
Despite the higher number of exclusive polymorphisms (22 in Sobral 1S and 16 in Sobral 2S) and the low number of shared polymorphisms (only six), no fixed differences were observed between the two groups. In the analysis of three allopatric populations of L. longipalpis from Brazil, Bauzer et al. (2002)
found a similar pattern and possible evidence of introgression. In the current article, only the 1S14br953 and 2S24br1027 sequences might indicate that this is happening between Sobral 1S and Sobral 2S. But it is also possible that they represent the persistence of an old polymorphism.
We calculated the pairwise estimates of population differentiation and gene flow between Sobral 1S and 2S and the populations of Jacobina, Lapinha, and Natal from Bauzer et al. (2002)
(see table 1
). The Fst and Nm values show that Sobral 2S is essentially the same gene pool as Natal, a locality situated more than 600 km away. This was confirmed in a tree including the Natal sequences (not shown), which shows a monophyletic group including sequences from both populations. This result is also in agreement with the pheromone data (Ward et al. 1988
; Souza et al. 2002
) and with recent preliminary copulation song analysis (Souza and Peixoto, unpublished data), which indicate that males from the two populations share the same Cembrene pheromone and produce similar copulation songs. On the other hand, Sobral 1S males are more closely related to Lapinha, with which they share the same one-spot phenotype and 9-methylgermacrene-B pheromone (Ward et al. 1988
; Hamilton et al. 1999b
; Hamilton and Ward, unpublished data) and to a lesser extent to Jacobina, a two-spot population with a different kind of pheromone, 3-methyl-
-himachalene (Hamilton et al. 1999a
). A neighbor-joining tree including sequences from these other two populations (not shown) reveals a more complex pattern with some alleles of Sobral 1S clustering with Jacobina and the rest clustering with Lapinha. Preliminary copulation song analysis (Souza and Peixoto, unpublished data) indicates that Sobral 1S males sing differently from Lapinha and Jacobina males, although the songs of these three populations are more similar in structure than the rather different songs observed in Natal and Sobral 2S males. Further molecular and behavioral work will help to determine the status of Sobral 1S relative to Lapinha and Jacobina.
Acknowledgements
We would like to thank Robson Costa da Silva for his excellent technical assistance, Jose Wellington and FUNASA (Sobral) for their help with the sandfly collections, and Karen Garner for her help with the DNA sequencing. This work was funded by The Wellcome Trust and UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) with additional support from Howard Hughes Medical Institute, Faperj, Fiocruz, The British Council, and CNPq.
Footnotes
Keywords: Lutzomyia longipalpis
period gene
sympatric species
visceral leishmaniasis
Address for correspondence and reprints: Alexandre A. Peixoto, Departamento de Bioquímica e Biologia Molecular, Fundação Oswaldo Cruz, Av. Brasil 4365, Manguinhos, CEP 21045-900 Rio de Janeiro, Brazil. E-mail: apeixoto{at}ioc.fiocruz.br
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