Molecular Characterization of Anopheles algeriensis Theobald, 1903 (Diptera: Culicidae) Populations from Europe

Abstract

Anopheles algeriensis Theobald, 1903, considered a competent vector of Plasmodium parasites, is a mosquito species widely distributed in the Mediterranean area but rare in Northern and Central Europe. The disappearance of its suitable breeding sites in Italy is having a detrimental effect on the occurrence of this species once common along the Southern coasts and on the islands. Recently, molecular investigations have renewed interest in this species, highlighting a genetic heterogeneity among European populations. In this study, An. algeriensis populations from Italy, Germany, Romania, and Sweden were analyzed by molecular typing of the intergenic transcribed spacer 2 (ITS2). The mitochondrial cytochrome c oxidase subunit I (COI) was also analyzed from specimens collected in Southern Italy. With the aim of investigating the population structure of this species, the obtained data were compared to all publicly available ITS2 and COI sequences of An. algeriensis, adding specimens from Spain and Portugal. The analyses of both markers indicate a split between Iberian populations (Spain for ITS2 and Spain/Portugal for COI) and those from the rest of Europe, revealing two cryptic species. The analysis of the COI barcode revealed a third clade representing a cryptic species present in Danube Delta (Romania). The high levels of genetic divergence among the clades of An. algeriensis indicate that this taxon represents a species complex, potentially harboring several distinct cryptic species.

Keywords: Anopheles algeriensis; barcoding; cryptic species; intergenic transcribed spacer 2; mitochondrial cytochrome c oxidase subunit I; phylogenetic analysis.

Figure 1

Map of sites of origin of Anopheles algeriensis specimens analyzed in this study. (1) Lesina (Foggia Province-Apulia); (2) Manfredonia (Foggia Province-Apulia); (3) Castellaneta (Taranto Province-Apulia); (4) Pisticci (Matera Province-Basilicata); (5) Policoro (Matera Province-Basilicata); (6) Klein Behnitz (Federal State of Brandenburg); (7) Wustrow (Federal State of Mecklenburg-Western Pomerania); (8) Sulina (Danube Delta Biosphere Reserve); (9) Lake Roşuleţ (Danube Delta Biosphere Reserve); (10) Gotland (Gotland Island); (11) Santiago do Cacém (Alenteyo Region); (12) Navarra Province; (13) Rioja Province; and (14) Majorca Island.

Figure 2

Phylogenetic and species delimitation analysis of An. algeriensis from Europe based on partial COI gene. Maximum clade credibility tree with numbers at branches indicating levels of support as Bayesian posterior probability >90%. An. superpictus (JX255708) and Culex pipiens biotype molestus (FN395171) were used as outgroup taxa. On the right are displayed the results of species delimitation analysis obtained by multi-rate Poisson tree process (mPTP), generalized mixed Yule coalescent model (GYMC), and the automatic barcode gap discoverer (ABGD). The Operational Taxonomic Units (OTUs) are also indicated.

Figure 3

Median-joining haplotype network based on partial COI gene representing populations of An. algeriensis from Europe analyzed in the present study. Each coloured circle represents a haplotype. The size of the circles is proportional to haplotype frequency. Hatch marks along the connecting edges represent substitutions differentiating two haplotypes. Small black circles represent median vectors, i.e., inferred (hypothetical or unsampled) sequences required to connect existing haplotypes. The clusters marked by the grey dashed lines are named (A–C), according to the designation based on species delimitation results (see Figure 2).

Figure 4

Phylogenetic and species delimitation analysis of An. algeriensis from Europe based on ITS2 sequences. Maximum clade credibility tree with numbers at branches indicating levels of support as Bayesian posterior probability >90%. An. superpictus (AY515163) was used as outgroup taxa. On the right are displayed the results of species delimitation analysis obtained by multi-rate Poisson tree process (mPTP) and the automatic barcode gap discoverer (ABGD). The Operational Taxonomic Units (OTUs) are also indicated.