**2. Distribution, biological aspects and population genetics of** *Lutzomyia umbratilis*

In the last years, as genetic molecular markers became available, the number of studies on population genetics and evolutionary genetics in sandfly species has significantly increased [44-49], and the results have revealed large intra-population genetic variation, genetically populations structured, genetic lineages and cryptic species complexes. In the case of vector species, the knowledge of the genetic structure of populations and the processes responsible for the differentiation distribution is important for the identification of the disease transmission heterogeneity patterns. Such patterns are often produced by the presence of cryptic species, structured populations and/or genetic lineages, which may show variation in the degrees of anthropophily, susceptibility of females to infection by the pathogen, infection rates and females longevity. The identification of these factors is of paramount importance for devel‐ oping effective management and vector control strategies.

The diversification patterns (structured populations, lineages, complete speciation) observed in sandfly species have generally been associated to multiple factors, such as climate barriers (or climate events in the past), geographic distances, differences in latitude or altitude, habitat modification, landscape fragmentation caused by anthropogenic actions and others, vegeta‐ tion type or geographic barriers (rivers, mountains). These factors can reduce the dispersal capacity of sandflies, leading them to become isolated populations and causing loss of genetic diversity and increase of differentiation among the populations, as discussed by Ready et al. [50] with regard to *Lutzomyia whitmani*, by Mukhopadhyay et al. [51] for *Lutzomyia shannoni*, by Uribe-Soto et al. [52] for *L. longipalpis* and by Pech-May et al. [45] for *Lutzomyia cruciata*. Additionally, the low flight capacity of this group of insects which seldom spread over more than 1 km, and the breeding soil type are also factors that may contribute even more to the population isolation and then favor the process of divergence and speciation events.

*Lutzomyiaumbratilis,* the main vector of *Le. guyanensi,* that causes Cutaneous Leishmaniasis (CL), occurs in northern South America, including Bolivia, Brazil, Colombia, French Guyana, Peru, Suriname and Venezuela [2,53]. In Brazil, *L. umbratilis* has been reported in all states of the northern region, the states of Mato Grosso and Mato Grosso do Sul (Southwest), besides the state of Maranhão and an isolated population in the state of Pernambuco, both in the northeastern region [2, 54,55]. Thus, populations of this species are spread over vast areas, separated by geographic barriers such as the largest rivers, the Amazon and the Negro, in the Brazilian Amazon region. Additionally, sandfly species have very limited dispersal capabili‐ ties, usually no more than 1 km [56,57], which favors geographic isolation of the populations. Thus, considering the vast geographic area, with discontinuous distribution, along with the low flight capacity of this insect group, *L. umbratilis* populations could be more susceptible to evolve into differentiated populations, incipient species and, ultimately, reproductively isolated species.

Similar to other insect groups, the Brazilian Amazon hosts a large diversity of sandfly species likely because of the great variety of ecological niches available [36] which are favorable for survival and reproduction. For example, in a single hectare of forest 50 sandfly species were captured [37]. This high level of diversity of insect vectors and also of reservoirs permits the simultaneous circulation of several species of *Leishmania* and is particularly interesting for the

In northern South America, in particular in the Brazilian Amazon region, the transmission of CL is associated to *Lutzomyia umbratilis* Ward and Fraiha and *Lutzomyia anduzei* Rozeboom, implicated as principal and secondary vectors of *Le. guyanensis*, respectively*.* The *Le. guyanen‐ sis* cycle is completed in several species of mammals, especially in xenarthrans, the two-toed sloth (*Choloepus didactylus*), considered the main reservoir, and marsupials such as the

**2. Distribution, biological aspects and population genetics of** *Lutzomyia*

In the last years, as genetic molecular markers became available, the number of studies on population genetics and evolutionary genetics in sandfly species has significantly increased [44-49], and the results have revealed large intra-population genetic variation, genetically populations structured, genetic lineages and cryptic species complexes. In the case of vector species, the knowledge of the genetic structure of populations and the processes responsible for the differentiation distribution is important for the identification of the disease transmission heterogeneity patterns. Such patterns are often produced by the presence of cryptic species, structured populations and/or genetic lineages, which may show variation in the degrees of anthropophily, susceptibility of females to infection by the pathogen, infection rates and females longevity. The identification of these factors is of paramount importance for devel‐

The diversification patterns (structured populations, lineages, complete speciation) observed in sandfly species have generally been associated to multiple factors, such as climate barriers (or climate events in the past), geographic distances, differences in latitude or altitude, habitat modification, landscape fragmentation caused by anthropogenic actions and others, vegeta‐ tion type or geographic barriers (rivers, mountains). These factors can reduce the dispersal capacity of sandflies, leading them to become isolated populations and causing loss of genetic diversity and increase of differentiation among the populations, as discussed by Ready et al. [50] with regard to *Lutzomyia whitmani*, by Mukhopadhyay et al. [51] for *Lutzomyia shannoni*, by Uribe-Soto et al. [52] for *L. longipalpis* and by Pech-May et al. [45] for *Lutzomyia cruciata*. Additionally, the low flight capacity of this group of insects which seldom spread over more than 1 km, and the breeding soil type are also factors that may contribute even more to the

population isolation and then favor the process of divergence and speciation events.

*Lutzomyiaumbratilis,* the main vector of *Le. guyanensi,* that causes Cutaneous Leishmaniasis (CL), occurs in northern South America, including Bolivia, Brazil, Colombia, French Guyana,

dynamic transmission studies of CL in this region [38,39].

opossum (*Didelphis marsupialis*) (*Didelphis marsupialis*) [40-43].

oping effective management and vector control strategies.

*umbratilis*

90 An Overview of Tropical Diseases

*Lutzomyia umbratilis* has been implicated in the transmission of *Le. guyanensis* in several countries of northern South America, including northern Brazil, and French Guiana and Suriname [58-60]. In the Brazilian Amazon, this species has shown to be highly anthropophilic and has been appointed as the main *Le. guyanensis* vector in the states of Pará [58-60], Amazonas [42,61-63] and Amapá [18] and is probably involved in the transmission in the states of Acre [64] and Rondônia [65]. Moreover, according to the hypothesis of Arias and Freitas [40], the susceptibility of this vector to *Leishmania* seems to vary in the central Brazilian Amazon region. *Lutzomyia umbratilis* populations naturally infected with *Le. guyanensis* have been observed east of the Negro River and north of the Amazonas River; however, there is no report of natural infections by *Leishmania* in this species south of the Amazon River system. Arias and Freitas [40] suggested that the fluvial system formed by the Amazon, Solimões and Negro Rivers may act as a barrier to the *Le. guyanensis* transmission cycle, where *L. umbratilis* populations display distinct degrees of vector competence between the opposite sides of these rivers, suggesting that these populations might represent a species complex or incipient speciation event.

Despite its importance as vector and the probable existence of a cryptic species complex, only few studies have tested the role of the rivers barrier in the genetic subdivision of *L. umbratilis*. A biological study conducted with two *L. umbratilis* populations from Manaus and Manaca‐ puru (left and right banks of the Negro River, respectively) in the Brazilian Amazon region, revealedsignificantdifferences in the life cycle,fecundity,fertility, emergencedegree andadult longevitybetweenthesepopulations,reflectingintrinsicbiologicaldifferences[66].Subsequent‐ ly, a study that combined morphology, chromosome and isozymes analyses of four *L. umbrati‐ lis* populations from this fluvial system showed significant differences in the bristle lengths of 4th instar larvae and in the number and size of the spines of the female genital atrium arma‐ ture[67].Thelatterhasbeenausefulmarkerfordistinguishingcloselyrelatedspeciesofsandflies [68]. Unfortunately, polytene chromosome analysis was not possible, but the metaphase karyotype was 2n=6. Isozymes did not reveal any differences among the populations [67]. This resultmaybeduetotheslowevolutionrate,negativeselectionandtheaminoacidcodonwobble effect. Consequently, isozymes are not informative markers for detecting incipient or recently diverged species. Therefore, the taxonomic status of *L. umbratilis* remains unclear.

*Lutzomyia umbratilis* was described by Ward and Fraiha [69], based on specimens captured in the Jari River region, state of Pará, Brazil. Because of the high morphological similarity between *L. umbratilis* and *L. anduzei*, the former has been wrongly identified as *L. anduzei* in the past. In fact, most of *L. anduzei* specimens found to be naturally infected with *Leishmania* before this date (1977) could actually be *L. umbratilis*. After this date, it has been possible distinguish *L. umbratilis* and *L. anduzei* morphologically, based on the internal and external genitalia of males and females [2,70]. Currently, they can also be identified molecularly by using DNA barcode sequences from *COI* of mitochondrial DNA [70]. The phylogenetic analysis of this dataset found two strongly supported monophyletic clades, although the genetic distances between them, based on the Kimura 2 Parameters (K2P) model, were very small (4.4%), suggesting that these species are very closely related (sister species) [70]. These species have been found infected naturally with *Le. guyanensis* in the Brazilian Amazon, although the studies revealed much higher infection rates in *L. umbratilis* than in *L. anduzei* females, consequently, the former has been recognized as principal vector of this parasite [18,63,71,72].

*L. umbratilis* adults are generally found in the rainforest (primary forest) of the Brazilian Amazon region, with its high humidity and dim light; therefore, the species has been recog‐ nized as ombrophilous, as expressed in its name, *L. umbratilis*. This species is further recognized as dendrobatic, because it is associated to tree trunks during the daytime. In the field, its density may vary, depending on the location and of these characteristics, but it seems to be denser in the central Amazon region, tending to reduce its density towards the edges of this region (Alencar, R. B., personal information). *L. umbratilis* adults are captured using aspirators on the bases of tree trunks during daytime and with CDC (Center for Disease Control) miniature light traps at ground level and in the forest canopy at night. These methods have been employed efficiently throughout Brazilian Amazon region [73].

In addition to the isozyme studies mentioned above, the most recent population genetics analyses were performed on the six *L. umbratilis* populations from the two opposite banks of the Amazon and Negro rivers (Table 2; Figure 3) by using a large fragment (1,181 bp) of the *COI* gene (the 3' end fragment of *COI*) [48] and the Barcode region (663 bp) [70], both from mitochondrial DNA. The aim of these analyses was to assess whether the populations of the opposite banks of these rivers consist of incipient or distinct species. In the study of Scarpassa and Alencar [48], 111 specimens were sequenced and the results revealed 52 haplotypes, reflecting a very large genetic variability for most of the samples examined, except one (Rio Preto da Eva). The genealogical relationships of the haplotypes were accessed using the TCS program [74] at the 95% confidence level. This analysis showed two haplotype groups (lineages), separated by ten mutational steps, but all connected in the network (Figure 4). Similarly, phylogenetic analysis using Bayesian Inference (BI) and inferred under the TIM1+I model, generated two distinct evolutionary lineages (probably clades), with probability support from moderate to slightly high (0.64 and 0.77; Figure 5), suggesting two monophyletic clades. These lineages can be separated by one fixed mutation at position 933 (A ↔ G) of the dataset, and the estimated sequence divergence between them was 1%. Lineage I consisted of four samples from the left bank of the Amazon and Negro rivers, whereas lineage II comprised two samples from the right bank of the Negro river (Figure 3). No haplotypes were shared between samples of the two lineages. Samples from the same clade (within-clades) exhibited low to moderate genetic differentiation (*F*ST = -0.0390-0.1841), whereas samples from different clades (between clades) exhibited extremely high and significant differentiation (*F*ST = 0.7100-0.8497; *P* < 0.0001) and fixed differences (*S*<sup>f</sup> = 1 to 7) (Table 3). Curiously, the samples from Manacapuru *versus* the samples from the BR-174 Highway, Rio Preto da Eva and Manaus, which are separated by smaller geographic distances (from 59.43 to 96.01 km), displayed more fixed differences (*S*<sup>f</sup> = 6 to 7) and no shared polymorphism (*S*<sup>s</sup> = 0), whereas, the samples from Manacapuru *versus* the samples from Cachoeira Porteira, which are separated by a larger geographic distance (449.22 km), exhibited less fixed differences (*S*<sup>f</sup> = 3) and more shared polymorphisms (*S*<sup>s</sup> = 2). Taken together, the evidence of absence of gene flow associated with the high levels of genetic differentiation may be an indicator of genetic discontinuity between these lineages, so they could represent incipient or distinct species. The separation time calculated between these lineages falls in the middle Pleistocene (0.22 Mya), coinciding with the more recent formation of the Amazon and Negro rivers [75], appointed as the most probable evolutionary force. This vicariant event, along with the low dispersal rate of the sandflies, and the amenable environmental conditions for adaptation and also drift are likely to have contributed to the great genetic differentiation between the populations of the opposite banks.

fact, most of *L. anduzei* specimens found to be naturally infected with *Leishmania* before this date (1977) could actually be *L. umbratilis*. After this date, it has been possible distinguish *L. umbratilis* and *L. anduzei* morphologically, based on the internal and external genitalia of males and females [2,70]. Currently, they can also be identified molecularly by using DNA barcode sequences from *COI* of mitochondrial DNA [70]. The phylogenetic analysis of this dataset found two strongly supported monophyletic clades, although the genetic distances between them, based on the Kimura 2 Parameters (K2P) model, were very small (4.4%), suggesting that these species are very closely related (sister species) [70]. These species have been found infected naturally with *Le. guyanensis* in the Brazilian Amazon, although the studies revealed much higher infection rates in *L. umbratilis* than in *L. anduzei* females, consequently, the former

*L. umbratilis* adults are generally found in the rainforest (primary forest) of the Brazilian Amazon region, with its high humidity and dim light; therefore, the species has been recog‐ nized as ombrophilous, as expressed in its name, *L. umbratilis*. This species is further recognized as dendrobatic, because it is associated to tree trunks during the daytime. In the field, its density may vary, depending on the location and of these characteristics, but it seems to be denser in the central Amazon region, tending to reduce its density towards the edges of this region (Alencar, R. B., personal information). *L. umbratilis* adults are captured using aspirators on the bases of tree trunks during daytime and with CDC (Center for Disease Control) miniature light traps at ground level and in the forest canopy at night. These methods have been employed

In addition to the isozyme studies mentioned above, the most recent population genetics analyses were performed on the six *L. umbratilis* populations from the two opposite banks of the Amazon and Negro rivers (Table 2; Figure 3) by using a large fragment (1,181 bp) of the *COI* gene (the 3' end fragment of *COI*) [48] and the Barcode region (663 bp) [70], both from mitochondrial DNA. The aim of these analyses was to assess whether the populations of the opposite banks of these rivers consist of incipient or distinct species. In the study of Scarpassa and Alencar [48], 111 specimens were sequenced and the results revealed 52 haplotypes, reflecting a very large genetic variability for most of the samples examined, except one (Rio Preto da Eva). The genealogical relationships of the haplotypes were accessed using the TCS program [74] at the 95% confidence level. This analysis showed two haplotype groups (lineages), separated by ten mutational steps, but all connected in the network (Figure 4). Similarly, phylogenetic analysis using Bayesian Inference (BI) and inferred under the TIM1+I model, generated two distinct evolutionary lineages (probably clades), with probability support from moderate to slightly high (0.64 and 0.77; Figure 5), suggesting two monophyletic clades. These lineages can be separated by one fixed mutation at position 933 (A ↔ G) of the dataset, and the estimated sequence divergence between them was 1%. Lineage I consisted of four samples from the left bank of the Amazon and Negro rivers, whereas lineage II comprised two samples from the right bank of the Negro river (Figure 3). No haplotypes were shared between samples of the two lineages. Samples from the same clade (within-clades) exhibited low to moderate genetic differentiation (*F*ST = -0.0390-0.1841), whereas samples from different clades (between clades) exhibited extremely high and significant differentiation (*F*ST =

has been recognized as principal vector of this parasite [18,63,71,72].

efficiently throughout Brazilian Amazon region [73].

92 An Overview of Tropical Diseases

**Figure 3.** Collection sites of *Lutzomyia umbratilis* from the Brazilian Amazon. Geographic distribution inferred of line‐ age I (in red color); Geographic distribution inferred of lineage II (in blue color). Map modified from Scarpassa and Alencar (2013) [70].


**Table 2.** Collection sites and sample sizes of *Lutzomyia umbratilis* from the Brazilian Amazon.

**Figure 4.** Parsimony haplotypes network of the 52 haplotypes observed in *Lutzomyia umbratilis*. H1 to H52, haplotypes. The haplotype circle sizes are proportional to number of individuals observed in each haplotype. Clade I is in red col‐ or. Clade II is in blue color. Empty smaller circles represent mutational events. **Source:** Scarpassa and Alencar (2012) [48].

Speciation in the *Leishmania guyanensis* Vector *Lutzomyia umbratilis* (Diptera: Psychodidae) from Northern Brazil… http://dx.doi.org/10.5772/60921 95

**Species Localities, State Co-ordinates N**

*L. umbratilis* Cachoeira Porteira, Oriximiná, Pará 1° 28' S; 56° 22' W 18

**Table 2.** Collection sites and sample sizes of *Lutzomyia umbratilis* from the Brazilian Amazon.

N: sample size. **Source:** Scarpassa and Alencar (2012) [48]

94 An Overview of Tropical Diseases

[48].

BR-174 Highway, Amazonas 2° 36' S; 60° 02' W 15 Rio Preto da Eva, Amazonas 2° 43' S; 59° 47' W 15 Manaus, Amazonas 3° 04' S; 59° 57' W 4 Manacapuru, Amazonas 3° 14' S; 60° 31' W 24 Novo Airão, Amazonas 2° 47' S; 60° 55' W 35

**Figure 4.** Parsimony haplotypes network of the 52 haplotypes observed in *Lutzomyia umbratilis*. H1 to H52, haplotypes. The haplotype circle sizes are proportional to number of individuals observed in each haplotype. Clade I is in red col‐ or. Clade II is in blue color. Empty smaller circles represent mutational events. **Source:** Scarpassa and Alencar (2012)

Latitude; Longitude

**Figure 5.** Bayesian Inference (BI) topology tree of the 52 haplotypes of *Lutzomyia umbratilis* inferred under the TIM1+I evolutionary model. Numbers above branch represent posterior probabilities obtained in the BI. *Lutzomyia anduzei* was used as outgroup. **Source:** Scarpassa and Alencar (2012) [48].


*F*ST: pair-wise genetic differentiation; *K:* average number of nucleotide differences between populations; *D*xy: average number of nucleotide substitutions per site between populations; *D*a: number of net nucleotide substitutions per site between populations; *S*s: number of shared polymorphisms between pairs of populations; *S*<sup>f</sup> : number of fixed differences between pairs of populations. The geographic distance (in km) between localities is represented inside the parentheses. \*\*\**P* = 0.00000 ± 0.0000, after the Bonferroni correction. **Source:** Scarpassa and Alencar (2012) [48].

**Table 3.** Genetic differentiation among samples and haplotype clade of *Lutzomyia umbratilis*.

Another study was conducted subsequently on these *L. umbratilis* populations, using the Barcode region (663bp) [70]. In the 72 specimens sequenced, 32 haplotypes were observed. In line with the results of the previous study [48], no haplotype was shared between lineages I and II. The genetic distance between the lineages, based on the K2P model, was rather small (0.009 to 0.010); however, they could be identified by one fixed mutation (T ↔ C transition at position 21).

The genetic differentiation observed in these studies supports the biological and morpholog‐ ical differences reported by Justiniano [67] and Justiniano et al. [66]. These results strongly indicate that *L. umbratilis* represents a species complex with recent evolutionary history. Taken together, these findings might explain possible differences in the vector competence of these sandflies, a hypothesis raised by Arias and Freitas [40]. On the other hand, these results do not support the isozyme data, which showed genetic homogeneity among populations. These inconsistencies between markers could be attributed to incomplete lineage sorting, due to recent divergence between *L. umbratilis* lineages (or distinct species) and/or distinct evolution rates of the markers used; for instance, isozymes evolve at a slower rate than mitochondrial DNA and are not informative markers for detecting incipient or recently diverged species.

Little is known about the natural breeding sites of *L. umbratilis* and, consequently, about its biology. This knowledge is important for application in any attempt to create and maintain colonies in laboratory conditions. The maintenance of *L. umbratilis* colonies could be the key to testing the mechanisms of reproductive isolation [66,76,77], as well as the assortative mating features between populations separated by the Negro and Amazon rivers, hypothesized as distinct species. It is particularly important because species that have diverged very recently are expected to share ancestral variation at high proportions, a situation that may confound their phylogenetic reconstruction. In addition, it is likely that in young species, with a recent divergence process, there are fixed differences only in genes involved in the speciation process. The maintenance of *L. umbratilis* colonies in the laboratory would also be important to assess the level of vector competence, based on tests of experimental infection between populations from the opposite river banks.

Another interesting approach could be genomic population studies using multilocus analysis, especially using loci which are involved in the different biologic aspects of *L. umbratilis*. This approach will permit distinguishing the effects of natural selection from those of genetic drift. The importance of this approach resides in the fact that genomic analyses provide more reliable information on historic and demographic events. The effect of a specific locus (outlier locus) helps identifying signs of natural selection in genes involved in the most variable adaptability process, such as those related to vector competence and (or) vector capacity, thus allowing a better understanding of vector status in distinct areas from the Brazilian Amazon.
