**3. Conclusion**

**Samples** *FST (Km) K Dxy Da Ss Sf*

Cachoeira Porteira x BR-174 Highway 0.0522 (368.40) 3.52 0.00297 0.00017 4 0

Cachoeira Porteira x Rio Preto da Eva 0.0569\*\*\* (353.67) 2.99 0.00248 0.00017 2 0

Cachoeira Porteira x Manaus 0.0230 (394.02) 3.92 0.00332 0.00025 1 0

BR-174 Highway x Rio Preto da Eva 0.0189 (30.46) 1.48 0.00125 0.00002 3 0

BR-174 Highway x Manaus -0.0390 (56.29) 2.20 0.00187 -0.00012 3 0

Rio Preto da Eva x Manaus 0.1841 (45.35) 1.87 0.00158 0.00008 2 0

Manacapuru x Novo Airão 0.0548 (58.74) 1.81 0.00153 0.00008 4 0

Cachoeira Porteira x **Manacapuru 0.7100\*\*\*** (449.22) 10.19 **0.00863 0.00599 2 3**

BR-174 Highway x **Manacapuru 0.8157\*\*\*** (87.11) 9.78 **0.00833 0.00673 0 6**

Rio Preto da Eva x **Manacapuru 0.8497\*\*\*** (96.01) 8.98 **0.00765 0.00653 0 6**

Manaus x **Manacapuru 0.8249\*\*\*** (59.43) 10.42 **0.00887 0.00699 0 7**

Cachoeira Porteira x **Novo Airão 0.7337 \*\*\*** (477.49) 10.55 **0.00899 0.00625 6 1**

BR-174 Highway x **Novo Airão 0.8197\*\*\*** (107.97) 10.21 **0.00869 0.00705 4 4**

Rio Preto da Eva x **Novo Airão 0.8439 \*\*\*** (130.14) 9.43 **0.00803 0.00687 1 4**

Manaus x **Novo Airão 0.8269\*\*\*** (108.76) 10.84 **0.00924 0.00731 2 5**

**Clade I** x **Clade II 0.7776\*\*\*** 9.99 **0.00850 0.00660 8 1**

between populations; *S*s: number of shared polymorphisms between pairs of populations; *S*<sup>f</sup>

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

position 21).

96 An Overview of Tropical Diseases

\*\*\**P* = 0.00000 ± 0.0000, after the Bonferroni correction. **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 pairs of populations. The geographic distance (in km) between localities is represented inside the parentheses.

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

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

: number of fixed differences

The two genetic lineages of *L. umbratilis* found in these studies may represent an advanced speciation process, indicating incipient or distinct species. This suggests that the Amazon and Negro rivers may be acting as effective barriers, as observed in *L. cruciata* [45], preventing gene flow between populations of opposite banks. Such findings have important implications for epidemiology, especially those related to vector competence, which are vital information for surveillance and vector control strategies in northern Brazil. Furthermore, this information may also provide a better knowledge of the evolutionary history of this species complex, as well as *L. umbratilis* represents an interesting example for speciation studies.

Finally, further studies of these populations using other molecular genetic markers, as well as additional sampling along the river banks and within interfluves in the Brazilian Amazon, are clearly needed to allow a more precise estimate of the differentiation, number of clades or distinct species. Studies of this kind are currently under way in our laboratory.
